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chitosan composite films
Food Hydrocolloids 97 (2019) 105208
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Effect of citric acid induced crosslinking on the structure and properties of potato starch/chitosan composite films
T
Hejun Wu∗,1, Yanlin Lei1, Junyu Lu, Rui Zhu, Di Xiao, Chun Jiao, Rui Xia, Zhiqing Zhang, Guanghui Shen, Yuntao Liu, Shanshan Li, Meiliang Li College of Food Science, Sichuan Agricultural University, No.46, Xin Kang Road, Yaan, Sichuan Province, 625014, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Potato starch Chitosan Citric acid Edible films Crosslinking Water resistance
Potato starch/chitosan (PS/CS) films cross-linked with citric acid (CA) at different concentrations (5%–20%, w/ w, on a dry basis of the weight of PS and CS) were developed via a solution blending-casting method, and their structure, water resistance, physical and mechanical properties, and antimicrobial activity were investigated. Fourier transform infrared spectra, X-ray diffraction and differential scanning calorimetry studies confirmed crosslinking among PS, CS and CA. Scanning electron microscopy images revealed that the cross-sectional fracture surfaces of all the films were smooth and homogenous, while the surface roughness of the CA crosslinked PS/CS films was higher than that of the uncross-linked films as also confirmed by the three-dimensional surface topography images. It was found that the properties of the films changed as the CA content varied, ascribing to the cosslinking and plasticizing effect of CA. The water resistance properties of the CA cross-linked PS/CS films were improved significantly when compared to the uncross-linked films. Moreover, the incorporation of CA could enhance the mechanical and antimicrobial properties of PS/CS films to some extent. Particularly, the results indicated that the films cross-linked with 15% CA showed the best comprehensive properties among all films. For example, the swelling degree of the films with 15% CA decreased from 686.4% to 98.1%, and water vapor permeability decreased from 3.03×10−12 g cm/cm2·s·Pa to 2.05 ×10−12 g cm/cm2·s·Pa, while the tensile strength was 29% higher than that of the uncross-linked film. However, excessive addition of CA in the composite films might solidify crystals on the film surface and have negative effects on their performance. This study provides a simple and effective pathway for preparation of polysaccharide-based films with improved properties, which have a potential as bioactive packaging material for food application.
1. Introduction In recent years, due to the concern about food safety and environmental issues caused by petroleum-based plastic packaging films, much attention have been focused on the development of edible and biodegradable films based on materials from renewable sources that are abundant in nature. The commonly investigated bio-based materials include polysaccharides, proteins and lipids (Cazón, Velazquez, Ramírez, & Vázquez, 2017; Gonzáleza, Gastelúa, Barrerad, Ribottad, & Igarzabal, 2019; Ren, Yan, Zhou, Tong, & Su, 2017). Among them, polysaccharide-based films are more attractive than protein-based and lipid-based films due to their advantages of low-cost, abundant resources, good film-forming ability as well as relatively stable performance (Carissimi, Flôres, & Rech, 2018). Starch is a polymer of D-glucose units linked by α-D-glycosidic
bonds, which consists of amylose and amylopectin molecules (Sujosh & Proshanta, 2018). This biopolymer has been extensively used as an attractive film-forming material for edible packaging in recent times due to its excellent film-forming properties and mechanical properties (Bonilla, Atarés, Vargas, & Chiralt, 2013; Niranjana & Prashantha, 2018). Nevertheless, the use of starch films for food packaging has been restricted due to their strong hydrophilic characteristic, such as poor moisture barrier properties and high water sensitivity (Niranjana & Prashantha, 2018). In this context, blending starch with other bio-based polymers (k-carrageenan, cellulose acetate, chitosan etc.) has been investigated in order to form composite materials with enhanced physical properties (Juliena, Mendietab, & Gutiérrezc, 2019; Mathew, Brahmakumar, & Abraham, 2010; Reddy & Yang, 2010; Shahbazi, Majzoobi, & Farahnaky, 2018; Zhong, Song, & Li, 2011). Chitosan (CS), a deacetylated derivative of chitin, is the second most
∗
Corresponding author. E-mail address: [email protected] (H. Wu). 1 These two authors contributed equally to this work. https://doi.org/10.1016/j.foodhyd.2019.105208 Received 27 March 2019; Received in revised form 1 July 2019; Accepted 1 July 2019 Available online 02 July 2019 0268-005X/ © 2019 Elsevier Ltd. All rights reserved.
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purpose of this work was to evaluate the effect of incorporating various concentration of citric acid on the structure, water resistance, thermal and physical properties, and antimicrobial activity of PS/CS films. The findings will provide the optimal formulation of PS/CS/CA composite films with suitable performances for potential food packaging application.
abundant polysaccharide found in the nature and mainly derived from shellfish processing waste (Ren et al., 2017). Various studies have demonstrated the possibility of development of edible films based on CS since it possesses excellent film-forming ability, selective permeability to gasses (CO2 and O2), good mechanical property as well as proven antimicrobial activity (Elsabee & Abdou, 2013; Sun et al., 2017). Chitosan has been previously added to films prepared with starches from different origins, such as potato (Mathew & Abraham, 2008), kudzu (Zhong et al., 2011), wheat (Bonilla et al., 2013), corn (Ren et al., 2017) and rice (Suriyatem, Auras, & Rachtanapun, 2018). Ren et al. (2017) developed composite films based on corn starch and chitosan, and reported that the composite films possessed better mechanical properties and water vapor permeability than films obtained from starch only, showing the potential to use as active packaging films in food and pharmaceutical field. Nevertheless, the water sensitivity of this sort of polysaccharide-based films still seems to be one of the main drawbacks, which might cause them to disintegrate when in contact with water, or to have their mechanical and barrier properties impaired because of water absorption and swelling, hindering many of their potential applications for food packaging purposes (Azeredo & Waldron, 2016; Sebti, Delves-Broughton, & Coma, 2003). Crosslinking is a promising technique to improve the performance and applicability of polysaccharide-based films, especially when concerning their water sensitivity (Aljawish et al., 2016; Azeredo & Waldron, 2016; Garavand, Rouhi, Razavi, Cacciotti, & Mohammadi, 2017; Li, Zhu, Guan, & Wu, 2019). Several crosslinking agents have been used for polysaccharides, including glutaraldehyde (Fan, Duquette, Dumont, & Simpson, 2018; Li, Gao, Wang, & Tong, 2013), ferulic acid (Li et al., 2019; Mathew & Abraham, 2008) and boric acid (Bahram et al., 2019). However, the actual application of those agents in the preparation of biomedical materials and films or coatings for various packaging material is limited due to their cytotoxicity, high cost and efficiency (Azeredo & Waldron, 2016; Li et al., 2019). For example, Li, Gao, Wang, Zhang, and Tong (2013) used glutaraldehyde as crosslinker to prepare chitosan/starch composite films. They found that the addition of glutaraldehyde had little effect on the water resistance properties of resulting films, and even led to poorer mechanical properties. Therefore, some effective and safe crosslinking agents for polysaccharide-based films are required to be better alternatives for food contact applications. Citric acid (CA), a bio-based polycarboxylic acid present in fruits, has been focused recently on their use as crosslinker owning to their low-cost and non-toxic nature, and ability to react and stabilize polysaccharide materials with high efficiency (García, Contreras, Hernández, & Palestino, 2017; Olsson, Hedenqvist, Johansson, & Järnström, 2013). The crosslinking mechanism is due to covalent intermolecular di-ester linkages between carboxyl groups of the crosslinking agents and hydroxyl groups of the polysaccharide (Azeredo et al., 2016). Several studies exploring the use of citric acid to improve various physical and barrier properties of polysaccharide-based films have been reported (Azeredo et al., 2016; Olsson et al., 2013; Priyadarshi, Sauraj, Kumar, & Negi, 2018; Seligra, Jaramillo, Lucía, & Goyanes, 2016). For example, in a study conducted by Olsson et al. (2013), it was found that thermoplastic starch films cross-linked with different amounts of citric acid had lower moisture content, diffusion coefficient and water vapor permeability values, which was attributed to the larger possibility to form hydrogen bonds between starch and citric acid. In another study, Priyadarshi, Sauraj Kumar, and Negi (2018) fabricated chitosan films incorporated with citric acid, which displayed better water resistance due to crosslinking as the values of moisture content, water absorption and water vapor permeability were decreased. Although citric acid has been previously employed as a cross-linking agent in single starch- or chitosan-based films, there are few studies conducted to investigate a combination of blending potato starch with chitosan and CA cross-linking to prepare composite films. Thus, the
2. Materials and methods 2.1. Materials Potato starch was purchased from Yuan Ye Biotechnology Co., Ltd. (Shanghai, China). Glycerol, Chitosan, citric acid and acetic acid were obtained from Chengdu Kelong Chemicals Co. Ltd. (Chengdu, China). Staphylococcus aureus (ATCC29213) and Escherichia coli (ATCC25922) were provided the College of Food Science, Sichuan Agricultural University. 2.2. Films preparation Potato starch was dispersed in deionized water to obtain 4% (w/v) starch solution (PS). The solution was heated at about 70 °C for 60 min under stirring to accomplish a complete starch gelatinization. Chitosan solution (CS, 1.5%, w/v) was prepared by dispersing chitosan in 0.5% (w/w) of acetic acid and stirring with a magnetic stirrer. The chitosan solution was blended with the potato starch solution (1:1, v/v). Then 15% glycerol (w/w, on a dry basis of the weight of PS and CS) was added and moderately stirred for 30 min at 40 °C. Citric acid (CA) was added at a concentration of 5%, 10%, 15%, 20% (w/w, on a dry basis of the weight of PS and CS) and the resulting dispersion was subjected to further mixing for 30 min, followed by degasification for 1 h using a vacuum pump. 200 mL solutions were cast on a 250 mm × 500 mm glass plate and dried at 55 °C for 6 h, and then the films were removed from the glass plate and conditioned at 23 ± 2 °C and 50% relative humidity for 48 h before running further tests. 2.3. Structural characterization 2.3.1. Fourier transform infrared (FTIR) analysis A Nicolet IS10 (Thermo Fisher Scientific, MA, USA) spectrometer with attenuated total reflectance (ATR) accessory was used to carry out FT-IR analysis. Measurements were obtained in the range of 500–4000 cm−1 as the average of 32 scans with a resolution of 4 cm−1. 2.3.2. X-ray diffraction (XRD) A Philips X-pert pro diffractometer (PANalytical, Holland)was used to observe the diffraction patterns of the PS/CS films with and without CA. X-ray generator tension and current were 40 kV and 40 mA, respectively. The diffraction patterns were obtained at room temperature over the 2θ range of 2–60° by step of 0.02°. 2.3.3. Scanning electron microscopy (SEM) The surfaces and the cross-sections of the films were observed by a ULTRA55 scanning electron microscope (Carl Zeiss AG, Germany) at a voltage of 10 kV. And the films were frozen in liquid nitrogen and then broken off into chips in order to observe the cross-sections. 2.3.4. Surface roughness observation The three-dimensional (3-D) VHX-1000C digital microscope (Keyence Corporation, Osaka, Japan) was used to evaluate the surface roughness of the films. The sample with a size of 50 mm × 30 mm was placed on the center of microscope stage with the machine direction from left to right. 2.3.5. Differential scanning calorimetry (DSC) Differential scanning calorimetry measurements were carried out 2
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and the thickness of films). And the opacity of the films was measured by UV–Vis 3100 spectrophotometer (Mapada Instruments, Shanghai, China) at a wavelength of 600 nm (Ren et al., 2017). Films were cut into to 10 mm×40 mm strips and put directly in a cuvette. And the opacity of film was calculated by the following equation:
using DSC 204 F1 (Netzsch, Germany). Samples of about 5 mg were weighed, encapsulated in aluminum pans individually and measured at a temperature range of −10 to 250 °C with a scan rate of 10 °C/min in a heating–cooling–heating cycle under a nitrogen flow. 2.3.6. Thermogravimetric analysis (TGA) Thermal analysis was performed on a TGA-Q 500 thermogravimetric analyzer (TA Instruments, USA). The films (10 mg) were weighed in alumina pans and heated from 30 °C to 800 °C with a constant heating rate of 10 °C/min under nitrogen atmosphere.
Opacity(mm−1) =
A600 X
(5)
Where A600 is the absorbance at 600 (nm) and X is the thickness of film (mm).
2.4. Water resistance properties
2.6. Mechanical properties
2.4.1. Moisture content (MC), water solubility (WS) and swelling degree (SD) The film pieces (50 mm × 50 mm) were prepared and weighed (W1), and then dried at 105 °C for 24 h and weighed again (W2). Then the dried samples were immersed into 40 mL distilled water at room temperature for 24 h. Undissolved film portions were filtered out, and the films were weighed after removal of the surface water with blotting paper (W3), then finally dried in an oven at 105 °C until a constant weight (W4) was reached. The MC (%), SD (%), and WS (%) values of the films were determined by the following equations (Zhang et al., 2019):
The tensile strength (TS) and elongation at break (EAB) of the films were determined with a TA.XI Plus Texture Analyzer (Super Technology Instruments Co., Ltd., China) according to the method of ASTM D882-18. Films were cut into strips (80 mm×15 mm) prior to analysis. The test speed was 60 mm/min, and the initial distance was 40 mm.
MC(%) =
W1 − W2 × 100% W1
2.7. Antimicrobial test The antimicrobial activity of the PS/CS films without and with CA against Staphylococcus aureus and Escherichia coli was evaluated by measuring the diameter of the disk inhibition zone. Broth cultures containing 106 CFU/mL of pathogens were prepared and spread onto culture medium. Film discs (14.2 mm in diameter) were placed onto the surface of agar plates containing respective culture medium. Then the prepared discs were incubated at 37 °C for 24 h, and the inhibition zones (diameter) were measured with a caliper and recorded in millimeters.
(1)
SD(%) =
W3 − W2 × 100% W2
(2)
WS(%) =
W2 − W4 × 100% W2
(3)
2.8. Statistical analysis 2.4.2. Water contact angle Multiple samples were tested and the data were presented as the average values ± standard deviation. The difference between factors and levels was evaluated by the analysis of variance (ANOVA) using SPSS 22.0 statistical analysis system. Duncan's multiple range tests were used to compare the means to identify which groups were significantly different from other groups (p < 0.05).
Water contact angles of the films were measured by an OCA20 contact angle analyzer (Dataphysics, Germany). A drop of deionized water (approximate 4 μL) was deposited onto the surface of the film, and the photo was taken at the same time. 2.4.3. Water vapor permeability (WVP) The WVP of the PS/CS composite films cross-linked without and with CA were measured by a PERME W3/031 WVP Tester (Lab think Instruments Co., Ltd. Jinan, China) according to the standard testing method of ASTM E96-16.
3. Results and discussion 3.1. Structural characterization 3.1.1. FTIR spectra FTIR was carried out to determine the possible functional chemical groups and molecular interactions of the cross-linked biopolymer films (Garavand et al., 2017). Fig. 1a showed the FT-IR spectra of CA and the PS/CS film cross-linked without and with CA. The spectrum of citric acid showed characteristic peaks at 3290 cm−1, 1745 cm−1 and 1698 cm−1, corresponding to O-H stretching and C=O stretching of carboxylic acid, respectively (Sampatrao et al., 2018). For the PS/CS film, the characteristic peak at a wavenumber of 3285 cm−1 was associated with the stretching vibration of the –OH group and the –NH2 group (Zhang et al., 2019). The position of the –OH group was almost consistent with the finding of Yusof, Shukur, Illias, and Kadir (2014) that appears at 3288 cm−1 with the blending of starch and chitosan. The absorption bands at 2926 cm−1 and 1595 cm−1 corresponded to CH stretching and N–H bending, respectively (Zhong et al., 2011). After adding CA into PS/CS film, a new peak appeared at 1724 cm−1, which could be attributed to the esterification between citric acid and either starch or chitosan, demonstrating the formation of chemical linkages among them (Reddy & Yang, 2010; Seligra et al., 2016; Shi et al., 2008). In addition, the peak at 1724 cm−1 intensified with increasing CA concentration, indicating the increased crosslinking
2.4.4. Moisture sorption Moisture sorption of the PS/CS/CA composite films was measured according to Gomaa's method with some modification (Gomaa, Hifney, Fawzy, & Gawad, 2018). The films were cut to 40 mm×40 mm strips, dried to a constant weight at 80 °C and placed in a desiccator maintained at 75% RH and 25 °C. The changes in the weight of the films were determined at fixed time intervals and the moisture sorption of the films was calculated as follows:
MS(%) =
Wt − W0 × 100% W0
(4)
Where Wo and Wt were the initial dry weight and the weight of the film sample at regular time, respectively. 2.5. Film thickness, density and opacity The film thickness was determined by taking ten measurements at different positions with a digital micrometer (Zhongtian Experimental Instrument Co. Ltd., Zhengzhou, China). Film density was calculated as the ratio of film weight to its volume (calculated according to the area 3
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Fig. 2. X-ray diffraction patterns of PS/CS films, PS/CS/CA 5% films, PS/CS/CA 15% films, and PS/CS/CA 20% films.
the composite films were obtained by X-ray diffraction (XRD) analysis. Fig. 2 showed XRD patterns of the PS/CS film cross-linked without and with CA. Characteristic peaks of PS/CS film at around 2θ = 17.14°, 19.76° and 22.14° could be attributed to the typical B-type crystals of starch and the regular crystal lattice of chitosan, which were in agreement with previous publications (Mathew, Brahmakumar, & Abraham, 2006; Ren et al., 2017; Suriyatem et al., 2018). When CA were incorporated into these two components, the intensity of characteristic XRD peaks decreased and some even disappeared, and particularly in the cross-linked films at 20% CA concentration, almost one single peak displayed at 17.55°, demonstrating that the intermolecular interactions between PS and CS limited the molecular chain segment movements, restrained their crystallization process and thus reduced the crystallinity of the matrix (Mathew et al., 2006; Ren et al., 2017; Shi et al., 2008; Zhong et al., 2011). The present result was in accordance with Reddy and Yang (2010) and Nataraj, Sakkara, Meghwal, and Reddy (2018), where the crystallinity of starch film and chitosan film decreased with the addition of CA, attributing to the crosslinking reaction caused by CA.
Fig. 1. FTIR spectra of CA and PS/CS films without and with CA (a) and the schematic illustration of possible structure of cross-linked system among CA, PS, and CS (b).
reaction. As indicated in Fig. 1b, the molecular interaction in the filmforming components was further illustrated in the form of a schematic diagram based on the results of the FT-IR spectra. Furthermore, the broad band around 3285 cm−1 became less intense when CA content increased to 15%, indicating the free O-H decreased due to the crosslinking interactions (esterification) (Li, Chen, Li, Bai, & Li, 2012; Seligra et al., 2016; Sun et al., 2017; Liu et al., 2017), as illustrated in Fig. 1b. However, this peak strengthened again for the films with 20% CA addition (Fig. 1a), probably due to excessive CA that unable to crosslink with the biopolymer matrix would cause an increase in the number of the hydroxyl and carboxyl groups and, thus, of the hydroxyl peak height (Shi et al., 2008). Similar observations were reported by Sun et al.(2014) in Chitosan films incorporated with gallic acid and Lei et al. (2019) in pectin-konjac glucomannan composite films incorporated with tea polyphenol, respectively, which indicated that the existence of excessive gallic acid or tea polyphenol would strengthen the peak around 3300 cm−1 (due to OH stretching). The results of ATR-FTIR suggested that crosslinking interactions occurred, which may subsequently affect the properties of PS/CS films.
3.1.3. Film morphology The microstructure and biopolymer compatibility of the developed films were analyzed by SEM, and micrographs of surface and crosssection of the films were shown in Fig. 3. Micrographs of the PS/CS film revealed smooth surfaces and compact structures without any noticeable pores or cracks, indicating the high compatibility and good filmforming properties of the two substances, regardless of whether crosslinking or not. This agreed with the findings of Bonilla et al. (2013) and Ren et al. (2017), who found a good molecular miscibility between starch and chitosan. However, it should be noted that, while the uncross-linked PS/CS film was relatively smooth, the films cross-linked with citric acid presented textured surfaces, particularly when a high concentration of CA (20%) was loaded, which might affect their physical properties. The starch-chitosan blend films with more compact structure and coarser surface due to the networking introduced by ferulic acid has also been reported by Mathew and Abraham (2008). On the other hand, the excessive citric acid crystals (marked by the red circles in Fig. 3) might solidify on sample with 20% CA, promoting a coarser surface on PS/CS films (Azeredo et al., 2015). Similarly, Liu et al. (2017) also observed large crystals on the surface of chitosan films when incorporating 1.5% or 2.0% of protocatechuic acid. VHX-1000C microscopic system was further employed to observe the surface roughness of the films and the resultant 3D surface
3.1.2. XRD The influence of crosslinking agents on the crystalline structure of 4
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Fig. 3. SEM micrographs of PS/CS films, PS/CS/CA 5% films, PS/CS/CA 15% films, and PS/CS/CA 20% films.
Fig. 4. 3D surface topography images of PS/CS films, PS/CS/CA 5% films, PS/CS/CA 15% films, and PS/CS/CA 20% films.
during the first heating cycle, in which all films showed one single endothermic peak. And as shown in Fig. 5b, the peak disappeared after the second heating cycle of the same sample, corresponding to water evaporation (Mathew et al., 2006). The maximum of this water evaporation peak became weaken and shifted from 82.3 to 122.1 °C when CA was incorporated into the PS/CS films, indicating the interactions of cross-linkages would hinder water evaporation (Mathew et al., 2006; Uranga et al., 2019). In order to further explore the thermal stability of the PS/CS/CA composite films, thermogravimetric analysis was utilized to analyze the structural interaction between polymers. The thermal degradation of CA and PS/CS films without and with CA as a function of temperature could be observed in Fig. 6. The weight loss at 40–150 °C could be attributed to the evaporation of weakly physically and strongly chemically bound water for all samples, as also shown by DSC in Fig. 5. It is
topography images were shown in Fig. 4. It can be observed from the 3D images that while the surface topographies of PS/CS blend films were relatively smooth and flat, the CA cross-linked PS/CS films had more protrusions than the uncross-linked films especially at high CA concentration, indicating the increased surface roughness (Gong et al., 2015; Xiong et al., 2014). The 3D surface topography image of PS/CS/ CA composite film was somewhat in agreement with those of ferulic acid incorporated starch-chitosan blend films studied by atomic force microscopy (Mathew & Abraham, 2008), which also exhibited a heterogenous surface with small patches of particles introduced by ferulic acid crosslinking. 3.1.4. Thermal analysis The DSC thermograms of PS/CS films cross-linked without and with CA were presented in Fig. 5. Fig. 5a is the DSC thermogram of the films 5
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Fig. 5. DSC thermograms of PS/CS films, PS/CS/CA 5% films, PS/CS/CA 15% films, and PS/CS/CA 20% films during the first (a) and second (b) heating cycles.
with 15% CA compared to the control, which could be indicative of stronger intermolecular interactions among the components (Li et al., 2019; Mathew et al., 2006), as previously confirmed by FTIR results. Similar results have also been found in starch-based film cross-linked with citric acid (Reddy & Yang, 2010). The structural characterization of PS/CS films preliminarily indicated the presence of crosslinking effect with CA addition, which could further enhance the interactions between molecules. These were further demonstrated by the water resistance, physical and mechanical properties of the films as given below.
worthwhile to mention that similar TG curve of CA in Fig. 6a has been reported by other authors (Thomas, Arun, Remya, & Nair, 2009). The major peak appeared around 212 °C was due to the decomposition of CA (Uranga et al., 2019). The thermograms of the composite films also showed a second stage of weight loss in the temperature range 200–400 °C and was caused by thermal degradation of the films, corresponding to complex processes including the dehydration of the saccharide rings, depolymerization and decomposition of the acetylated and deacetylated units of polymer (Mathew & Abraham, 2008; Mathew et al., 2006). No significant difference in the thermal stability of the control and citric acid incorporated films could be observed from TGA curves in Fig. 5. The stages of weight loss can be investigated better in the DTG curves. The maximum temperature corresponding to each stage seemed to appear at higher temperatures when CA was incorporated into the films, increasing by ∼10 °C and 5 °C for the films
3.2. Water resistance properties 3.2.1. Moisture content, water solubility and swelling degree Moisture content was determined to evaluate the amount of
Fig. 6. TGA and DTG curves of CA, PS/CS films, PS/CS/CA 5% films, and PS/CS/CA 15% films. 6
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Table 1 Moisture content (MC), water solubility (WS), swelling degree (SD) and water contact angle (WCA) of PS/CS films without and with CA. CA
MC
WS
SD
WCA
(w/w)
(%)
(%)
(%)
(°)
0%
17.91 ± 1.11a
50.65 ± 2.15a
686.4 ± 39.6a
5%
17.47 ± 0.29 ab
28.63 ± 1.34b
346.8 ± 26.1b
10%
16.13 ± 0.77bc
24.35 ± 0.66c
184.9 ± 3.1c
15%
16.01 ± 1.20bc
21.60 ± 1.00d
98.1 ± 8.8e
20%
11.14 ± 0.44d
26.41 ± 0.84bc
143.3 ± 4.6d
Values are presented as mean ± standard deviation. Different letters in the same column indicate significant differences (p < 0.05).
moisture in the film. As can be seen from Table 1, the moisture content of the PS/CS films gradually decreased as the increasing of CA content, ranging from 11.14% to 17.91%, in agreement with the weight loss associated to the first step of TGA curves (Fig. 6). Some other studies reported similar findings of films cross-linked with CA (Olsson et al., 2013; Seligra et al., 2016). The decrease of MC in PS/CS/CA films supported the proposal about crosslinking and the consequent decrease of free hydrophilic groups in the films, as previously illustrated in Fig. 1. Water solubility of PS/CS films cross-linked without and with CA was presented in Table 1. The results of WS, decreasing from 50.65 ± 0.02% for control PS/CS films to 21.60 ± 0.08% for PS/CS/ CA 15% films, indicated that the films were less soluble by adding CA. This may be due to the decreased availability of hydrophilic hydroxyl groups and generated hydrophobic ester groups between citric acid and the polysaccharides, resulting in a denser structure and corresponding improved water insolubility of PS/CS/CA compared to films without CA (Reddy & Yang, 2010; Garavand et al., 2017). However, the WS of PS/ CS/CA films increased again when added 20% CA, probably ascribed to the extra-added CA that unable to be cross-linked with polymers, thus leading to more hydroxyl groups and attracting water (Ghanbarzadeh, Almasi, & Entezami, 2011). Swelling degree of PS/CS films cross-linked without and with CA was also presented in Table 1. Cross-linking can cause the films to become more insoluble due to chemical interactions between the chitosan and starch molecules (Li et al., 2013). It could be observed that the SD of the PS/CS/CA films was a significantly lower than that of the PS/CS films (p < 0.05), indicating a lower free volume between knots and a higher crosslinking interactions between PS and CS, which reduced the number of hydrogen bonds to water and created a more dense structure with better resistance (Reddy & Yang, 2010; Suriyatem et al., 2018). In particular, SD of the PS/CS/CA 15% films significantly decreased from 686.4% to 98.1%.
3.2.2. Water contact angle Water contact angle (WCA) was determined to evaluate the hydrophobic nature of the films as well as wettability of their surface. Table 1 showed the WCA values of the PS/CS films cross-linked without and with CA. The WCA of the PS/CS films increased from 34.47° to 56.43° (with addition of 15% CA), indicating that the surface hydrophobicity of PS/CS films was markedly improved. This could be related to the formation of hydrophobic ester groups between citric acid and the polysaccharides, leading to a decrease in the number of polar groups as shown by FTIR analysis (Fig. 1), thus hindering the water absorption from the film surface (Uranga et al., 2019). Moreover, it has been reported that the rough surfaces may have substantially larger contact angles due to heterogeneous wetting (Xiong et al., 2014). The increased surface roughness of CA crosslinked films, evident in the SEM and 3D surface topography images (Figs. 3 and 4), could probably result in larger WCA values. However, WCA value decreased to 51.73° when incorporated with 20% CA, possibly due to the free hydrophilic groups (-COOH and -OH) from excessive addition of CA which solidified crystals on the films’ surface (as confirmed by FTIR spectra in Fig. 1 and shown in Fig. 3). Similar changes of WCA in polysaccharide-based films carrying tea polyphenol were also observed by other researchers (Feng et al., 2018; Lei et al., 2019). 3.2.3. Water vapor permeability Fig. 7a showed the WVP values of PS/CS films cross-linked without and with CA. The WVP of the PS/CS film (3.03 ×10−12 g cm/cm2·s·Pa) was close to the values for corn starch/chitosan films in previous reports (1.22×10−12–3.04×10−12 g cm/cm2·s·Pa) by Ren et al. (2017). The WVP of the CA cross-linked PS/CS films was significantly lower than the WVP of the PS/CS film (p < 0.05), ranging from 2.05 ×10−12 g cm/cm2·s·Pa to 2.81 ×10−12 g cm/cm2·s·Pa. It had been reported that the WVP of a film depended on the number of available polar groups that the polymer contains. The decreased WVP values when added CA might be ascribed to the replacement of hydrophilic hydroxyl groups with hydrophobic ester groups (Garavand et al., 2017). 7
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Fig. 7. Water vapor permeability (a) and moisture sorption curve (b) of PS/CS films without and with CA.
amount of solid content in film-forming solution and thus increased the film thickness. Moreover, with the increase in CA concentration, the crosslinking interactions would cause tighter binding between polymers, resulting in more compact film structure and thereby increasing the density of the PS/CS/CA films (Sun et al., 2017; Wu et al.,. 2019). Table 2 also showed the opacity values as a function of CA concentration on the PS/CS films. Generally, the greater the opacity value, the less transparent were the films. It could be observed that the PS/CS/CA films were less transparent (with higher opacity value) than those of PS/CS films. A former study reported by Li et al. (2019) corroborated the above-mentioned changes in opacity, ascribed to the reaction between crosslinker (CA) and matrix, resulting in a tougher surface (evident in Figs. 3 and 4) and corresponding higher opacity. Nevertheless, it is noteworthy that the composite films still showed good light transmittance that could favor consumer to see the products when used as packaging materials.
On the other hand, the cross-linking effect after CA addition, led to a denser film structure with strong interfacial interaction, which was also beneficial to improve water barrier ability (Garavand et al., 2017; Lei et al., 2019). Seligra et al. (2016) also reported a decrease in the WVP values of starch–glycerol films, when adding CA as crosslinking agent. However, the WVP of PS/CS/CA films increased when added 20% CA. This could be probably explained by the plasticizing role of excessive CA addition, which could increase the mobility of the inter-chain and chain spaces, leading to increasing film diffusivity and high speed of water vapor transmission (Garavand et al., 2017; Ghanbarzadeh et al., 2011). Similar changing tendency in WVP have been reported by Mathew and Abraham (2008) for ferulic acid incorporated starchchitosan blend films and Ghanbarzadeh et al. (2011) for citric acid incorporated starch films. 3.2.4. Moisture sorption The results of the moisture sorption as a function of time of the PS/ CS films without and with CA were depicted in Fig. 7b. It could be observed that the moisture sorption of PS/CS and PS/CS/CA composite films increased with increasing time and reached balance at the 40th hour. And the moisture sorption rate at the initial stages was more rapid and declined with increasing time. Similar results were also obtained by Gomaa, Hifney, Fawzy, and Abdel-Gawad (2018). Moisture sorption of the PS/CS composite films decreased with the increase of CA content, reaching a minimal MS equilibrium value of ∼10.1% for films with 15% CA, and increased again when 20% CA was incorporated. As mentioned above, the supposed reason can be explained by the fact that cross-linking effect of CA resulted in hydrophobic films with denser structure, and thereby improved their water resistance. Whereas, the presence of numerous hydrophilic hydroxyl groups from extra-added CA might tend to interact with water molecules (Garavand et al., 2017), as evidenced by the results of FTIR and WCA.
3.3.2. Mechanical properties The effects of different concentrations of CA on the mechanical properties of PS/CS films were presented in Table 2. The presence of CA caused significant (p < 0.05) differences in the TS and EAB. PS/CS films had relatively lower mechanical properties, TS and EAB of which were 9.72 ± 0.87 MPa and 5.70 ± 0.01%, respectively. Generally, it had been reported that the incorporation of increasing amount of crosslinkers led to improved TS and decreased EAB of biopolymer films (Garavand et al., 2017). As seen from Table 2, TS of the PS/CS composite films increased with increasing of CA concentration, and reached a maximum (29% higher than uncross-linked film) at 15% CA concentration, then slightly declined at 20% CA concentration. While EAB increased with increasing of CA concentration. As seen from Table 1, since the moisture content of the CA crosslinked PS/CS films was lower than the uncrosslinked films, the change in mechanical properties can not be attributed to the plasticizing effect of water. The improved TS obtained by the addition of CA could be due to intra and inter-molecular crosslinking among PS, CS, and CA (as illustrated in Fig. 1b). This interaction contributed to a more compact structure and thereby improved TS of the PS/CS composite films (Mathew & Abraham, 2008). On the other hand, CA may act both as a crosslinking agent and a plasticizer in the biopolymer films, especially at high contents. The extra (not-reacted) portion of citric acid probably acts as a plasticizer and reduces the interactions between the macromolecules, with consequent TS decrement and EAB increase (Garavand et al., 2017; Wang, Ren, Li, Sun, & Liu, 2014). Comparable results were reported by Mathew & Abraham, (2008) and Wang et al. (2014). Wang
3.3. Physical and mechanical properties 3.3.1. Thickness, density and opacity As seen from Table 2, the incorporation of CA promoted an increase in film thickness and density in comparison with the control PS/CS film, and this augmentation was more pronounced in the films with higher CA content (p < 0.05). The thicknesses of the films is directly proportional to the concentration of solids in the formulation, and can be controlled by weighing samples of the blended solution to the same mass prior to casting onto the 250 mm × 500 mm glass plate (Li et al., 2013). The presence of citric acid would have contributed to the more 8
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Table 2 Physical and mechanical properties of PS/CS films without and with CA. CA (w/w) 0% 5% 10% 15% 20%
Thickness (μm) 76.67 80.00 81.33 82.67 86.00
Density
Opacity
−3
± ± ± ± ±
1.53c 1.00b 2.08b 1.53b 1.73a
(g·cm 1.243 1.248 1.251 1.452 1.663
) ± ± ± ± ±
−1
(mm ) 0.92 ± 0.11c 0.98 ± 0.02c 1.04 ± 0.09bc 1.34 ± 0.02b 1.98 ± 0.10a
0.02c 0.01c 0.03c 0.02b 0.05a
TS
EAB
(MPa) 9.72 ± 0.87b 9.27 ± 0.09b 12.53 ± 0.84a 12.55 ± 0.97a 10.49 ± 0.38b
(%) 5.70 6.39 7.13 7.49 8.96
± ± ± ± ±
0.1d 0.19c 0.01b 0.41b 0.62a
Values are presented as mean ± standard deviation. Different letters in the same column indicate significant differences (p < 0.05).
Conflict of interest statement
Table 3 Antimicrobial properties of PS/CS films without and with CA. CA
Inhibition zone diameter (mm)
(w/w) 0% 5% 10% 15% 20%
E. coli (-) 16.48 ± 0.13c 17.09 ± 0.41b 17.14 ± 0.23b 17.32 ± 0.39b 18.14 ± 0.12a
The authors declare no conflict of interest in the publication of this article. S. aureus (+) 16.32 ± 0.33c 17.17 ± 0.64bc 17.99 ± 1.45 ab 18.23 ± 0.70 ab 18.93 ± 0.62a
Acknowledgments This work was supported by Seedling Project of Sichuan Science and Technology Department (2018126), General Program of Natural Science of Sichuan Education Department (16ZB0057).
Values are presented as mean ± standard deviation. Different letters in the same column indicate significant differences (p < 0.05).
Appendix A. Supplementary data
et al. (2014) investigated the effect of citric acid on the mechanical behavior of polyvinyl alcohol/xylan composite films. And they observed that films with 10 wt% citric acid increased TS, when compared to the control film, evidencing a crosslinking effect. However, citric acid concentrations higher than 10 wt% caused the TS to decrease and the EAB to increase, suggesting a plasticizing effect. On the basis of all the collected data, it can be concluded that incorporation of CA as a crosslinking agent in the desired range can modify the properties of PS/CS films, otherwise the excess amount acts as a plasticizer and has an unfavorable impact.
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3.4. Antimicrobial properties The inhibitory effects of PS/CS/CA films on the growth of the two bacteria including the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli bacteria were summarized in Table 3. As could be observed from Table 3, the control PS/CS films showed some inhibitory effect on Escherichia coli and Staphylococcus aureus, which was due to the antimicrobial nature of chitosan (Nataraj et al., 2018). And the diameter of inhibition zones increased as the concentration of CA increased, which was ascribed to the fact that CA has inhibitory effects against some bacteria (Mahmoud, 2014). The PS/CS/CA films with antibacterial properties indicated that they would be beneficial for its practical application in the future. 3. Conclusions PS and CS blend films cross-linked without and with CA were developed and their structural, physical and antimicrobial properties were characterized. The FTIR, XRD and DSC analysis indicated the CA crosslinking interactions occurred between PS and CS. The PS/CS/CA films exhibited a homogenous and denser structure with higher surface roughness as well as enhanced water resistance, mechanical and antimicrobial properties compared with uncross-linked films. Particularly, the results indicated that the films cross-linked with 15% CA showed the best comprehensive properties among all films. However, overloading of CA (20%) would slightly decrease mechanical and water resistance properties, possibly due to the plasticizing role of CA. This study provided the appropriate formula of developing CA crosslinked PS/CS films with desirable properties that possess potential in food preservation and packaging industry. 9
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Effect of citric acid induced crosslinking on the structure and properties of potato starch/chitosan composite films
T
Hejun Wu∗,1, Yanlin Lei1, Junyu Lu, Rui Zhu, Di Xiao, Chun Jiao, Rui Xia, Zhiqing Zhang, Guanghui Shen, Yuntao Liu, Shanshan Li, Meiliang Li College of Food Science, Sichuan Agricultural University, No.46, Xin Kang Road, Yaan, Sichuan Province, 625014, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Potato starch Chitosan Citric acid Edible films Crosslinking Water resistance
Potato starch/chitosan (PS/CS) films cross-linked with citric acid (CA) at different concentrations (5%–20%, w/ w, on a dry basis of the weight of PS and CS) were developed via a solution blending-casting method, and their structure, water resistance, physical and mechanical properties, and antimicrobial activity were investigated. Fourier transform infrared spectra, X-ray diffraction and differential scanning calorimetry studies confirmed crosslinking among PS, CS and CA. Scanning electron microscopy images revealed that the cross-sectional fracture surfaces of all the films were smooth and homogenous, while the surface roughness of the CA crosslinked PS/CS films was higher than that of the uncross-linked films as also confirmed by the three-dimensional surface topography images. It was found that the properties of the films changed as the CA content varied, ascribing to the cosslinking and plasticizing effect of CA. The water resistance properties of the CA cross-linked PS/CS films were improved significantly when compared to the uncross-linked films. Moreover, the incorporation of CA could enhance the mechanical and antimicrobial properties of PS/CS films to some extent. Particularly, the results indicated that the films cross-linked with 15% CA showed the best comprehensive properties among all films. For example, the swelling degree of the films with 15% CA decreased from 686.4% to 98.1%, and water vapor permeability decreased from 3.03×10−12 g cm/cm2·s·Pa to 2.05 ×10−12 g cm/cm2·s·Pa, while the tensile strength was 29% higher than that of the uncross-linked film. However, excessive addition of CA in the composite films might solidify crystals on the film surface and have negative effects on their performance. This study provides a simple and effective pathway for preparation of polysaccharide-based films with improved properties, which have a potential as bioactive packaging material for food application.
1. Introduction In recent years, due to the concern about food safety and environmental issues caused by petroleum-based plastic packaging films, much attention have been focused on the development of edible and biodegradable films based on materials from renewable sources that are abundant in nature. The commonly investigated bio-based materials include polysaccharides, proteins and lipids (Cazón, Velazquez, Ramírez, & Vázquez, 2017; Gonzáleza, Gastelúa, Barrerad, Ribottad, & Igarzabal, 2019; Ren, Yan, Zhou, Tong, & Su, 2017). Among them, polysaccharide-based films are more attractive than protein-based and lipid-based films due to their advantages of low-cost, abundant resources, good film-forming ability as well as relatively stable performance (Carissimi, Flôres, & Rech, 2018). Starch is a polymer of D-glucose units linked by α-D-glycosidic
bonds, which consists of amylose and amylopectin molecules (Sujosh & Proshanta, 2018). This biopolymer has been extensively used as an attractive film-forming material for edible packaging in recent times due to its excellent film-forming properties and mechanical properties (Bonilla, Atarés, Vargas, & Chiralt, 2013; Niranjana & Prashantha, 2018). Nevertheless, the use of starch films for food packaging has been restricted due to their strong hydrophilic characteristic, such as poor moisture barrier properties and high water sensitivity (Niranjana & Prashantha, 2018). In this context, blending starch with other bio-based polymers (k-carrageenan, cellulose acetate, chitosan etc.) has been investigated in order to form composite materials with enhanced physical properties (Juliena, Mendietab, & Gutiérrezc, 2019; Mathew, Brahmakumar, & Abraham, 2010; Reddy & Yang, 2010; Shahbazi, Majzoobi, & Farahnaky, 2018; Zhong, Song, & Li, 2011). Chitosan (CS), a deacetylated derivative of chitin, is the second most
∗
Corresponding author. E-mail address: [email protected] (H. Wu). 1 These two authors contributed equally to this work. https://doi.org/10.1016/j.foodhyd.2019.105208 Received 27 March 2019; Received in revised form 1 July 2019; Accepted 1 July 2019 Available online 02 July 2019 0268-005X/ © 2019 Elsevier Ltd. All rights reserved.
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purpose of this work was to evaluate the effect of incorporating various concentration of citric acid on the structure, water resistance, thermal and physical properties, and antimicrobial activity of PS/CS films. The findings will provide the optimal formulation of PS/CS/CA composite films with suitable performances for potential food packaging application.
abundant polysaccharide found in the nature and mainly derived from shellfish processing waste (Ren et al., 2017). Various studies have demonstrated the possibility of development of edible films based on CS since it possesses excellent film-forming ability, selective permeability to gasses (CO2 and O2), good mechanical property as well as proven antimicrobial activity (Elsabee & Abdou, 2013; Sun et al., 2017). Chitosan has been previously added to films prepared with starches from different origins, such as potato (Mathew & Abraham, 2008), kudzu (Zhong et al., 2011), wheat (Bonilla et al., 2013), corn (Ren et al., 2017) and rice (Suriyatem, Auras, & Rachtanapun, 2018). Ren et al. (2017) developed composite films based on corn starch and chitosan, and reported that the composite films possessed better mechanical properties and water vapor permeability than films obtained from starch only, showing the potential to use as active packaging films in food and pharmaceutical field. Nevertheless, the water sensitivity of this sort of polysaccharide-based films still seems to be one of the main drawbacks, which might cause them to disintegrate when in contact with water, or to have their mechanical and barrier properties impaired because of water absorption and swelling, hindering many of their potential applications for food packaging purposes (Azeredo & Waldron, 2016; Sebti, Delves-Broughton, & Coma, 2003). Crosslinking is a promising technique to improve the performance and applicability of polysaccharide-based films, especially when concerning their water sensitivity (Aljawish et al., 2016; Azeredo & Waldron, 2016; Garavand, Rouhi, Razavi, Cacciotti, & Mohammadi, 2017; Li, Zhu, Guan, & Wu, 2019). Several crosslinking agents have been used for polysaccharides, including glutaraldehyde (Fan, Duquette, Dumont, & Simpson, 2018; Li, Gao, Wang, & Tong, 2013), ferulic acid (Li et al., 2019; Mathew & Abraham, 2008) and boric acid (Bahram et al., 2019). However, the actual application of those agents in the preparation of biomedical materials and films or coatings for various packaging material is limited due to their cytotoxicity, high cost and efficiency (Azeredo & Waldron, 2016; Li et al., 2019). For example, Li, Gao, Wang, Zhang, and Tong (2013) used glutaraldehyde as crosslinker to prepare chitosan/starch composite films. They found that the addition of glutaraldehyde had little effect on the water resistance properties of resulting films, and even led to poorer mechanical properties. Therefore, some effective and safe crosslinking agents for polysaccharide-based films are required to be better alternatives for food contact applications. Citric acid (CA), a bio-based polycarboxylic acid present in fruits, has been focused recently on their use as crosslinker owning to their low-cost and non-toxic nature, and ability to react and stabilize polysaccharide materials with high efficiency (García, Contreras, Hernández, & Palestino, 2017; Olsson, Hedenqvist, Johansson, & Järnström, 2013). The crosslinking mechanism is due to covalent intermolecular di-ester linkages between carboxyl groups of the crosslinking agents and hydroxyl groups of the polysaccharide (Azeredo et al., 2016). Several studies exploring the use of citric acid to improve various physical and barrier properties of polysaccharide-based films have been reported (Azeredo et al., 2016; Olsson et al., 2013; Priyadarshi, Sauraj, Kumar, & Negi, 2018; Seligra, Jaramillo, Lucía, & Goyanes, 2016). For example, in a study conducted by Olsson et al. (2013), it was found that thermoplastic starch films cross-linked with different amounts of citric acid had lower moisture content, diffusion coefficient and water vapor permeability values, which was attributed to the larger possibility to form hydrogen bonds between starch and citric acid. In another study, Priyadarshi, Sauraj Kumar, and Negi (2018) fabricated chitosan films incorporated with citric acid, which displayed better water resistance due to crosslinking as the values of moisture content, water absorption and water vapor permeability were decreased. Although citric acid has been previously employed as a cross-linking agent in single starch- or chitosan-based films, there are few studies conducted to investigate a combination of blending potato starch with chitosan and CA cross-linking to prepare composite films. Thus, the
2. Materials and methods 2.1. Materials Potato starch was purchased from Yuan Ye Biotechnology Co., Ltd. (Shanghai, China). Glycerol, Chitosan, citric acid and acetic acid were obtained from Chengdu Kelong Chemicals Co. Ltd. (Chengdu, China). Staphylococcus aureus (ATCC29213) and Escherichia coli (ATCC25922) were provided the College of Food Science, Sichuan Agricultural University. 2.2. Films preparation Potato starch was dispersed in deionized water to obtain 4% (w/v) starch solution (PS). The solution was heated at about 70 °C for 60 min under stirring to accomplish a complete starch gelatinization. Chitosan solution (CS, 1.5%, w/v) was prepared by dispersing chitosan in 0.5% (w/w) of acetic acid and stirring with a magnetic stirrer. The chitosan solution was blended with the potato starch solution (1:1, v/v). Then 15% glycerol (w/w, on a dry basis of the weight of PS and CS) was added and moderately stirred for 30 min at 40 °C. Citric acid (CA) was added at a concentration of 5%, 10%, 15%, 20% (w/w, on a dry basis of the weight of PS and CS) and the resulting dispersion was subjected to further mixing for 30 min, followed by degasification for 1 h using a vacuum pump. 200 mL solutions were cast on a 250 mm × 500 mm glass plate and dried at 55 °C for 6 h, and then the films were removed from the glass plate and conditioned at 23 ± 2 °C and 50% relative humidity for 48 h before running further tests. 2.3. Structural characterization 2.3.1. Fourier transform infrared (FTIR) analysis A Nicolet IS10 (Thermo Fisher Scientific, MA, USA) spectrometer with attenuated total reflectance (ATR) accessory was used to carry out FT-IR analysis. Measurements were obtained in the range of 500–4000 cm−1 as the average of 32 scans with a resolution of 4 cm−1. 2.3.2. X-ray diffraction (XRD) A Philips X-pert pro diffractometer (PANalytical, Holland)was used to observe the diffraction patterns of the PS/CS films with and without CA. X-ray generator tension and current were 40 kV and 40 mA, respectively. The diffraction patterns were obtained at room temperature over the 2θ range of 2–60° by step of 0.02°. 2.3.3. Scanning electron microscopy (SEM) The surfaces and the cross-sections of the films were observed by a ULTRA55 scanning electron microscope (Carl Zeiss AG, Germany) at a voltage of 10 kV. And the films were frozen in liquid nitrogen and then broken off into chips in order to observe the cross-sections. 2.3.4. Surface roughness observation The three-dimensional (3-D) VHX-1000C digital microscope (Keyence Corporation, Osaka, Japan) was used to evaluate the surface roughness of the films. The sample with a size of 50 mm × 30 mm was placed on the center of microscope stage with the machine direction from left to right. 2.3.5. Differential scanning calorimetry (DSC) Differential scanning calorimetry measurements were carried out 2
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and the thickness of films). And the opacity of the films was measured by UV–Vis 3100 spectrophotometer (Mapada Instruments, Shanghai, China) at a wavelength of 600 nm (Ren et al., 2017). Films were cut into to 10 mm×40 mm strips and put directly in a cuvette. And the opacity of film was calculated by the following equation:
using DSC 204 F1 (Netzsch, Germany). Samples of about 5 mg were weighed, encapsulated in aluminum pans individually and measured at a temperature range of −10 to 250 °C with a scan rate of 10 °C/min in a heating–cooling–heating cycle under a nitrogen flow. 2.3.6. Thermogravimetric analysis (TGA) Thermal analysis was performed on a TGA-Q 500 thermogravimetric analyzer (TA Instruments, USA). The films (10 mg) were weighed in alumina pans and heated from 30 °C to 800 °C with a constant heating rate of 10 °C/min under nitrogen atmosphere.
Opacity(mm−1) =
A600 X
(5)
Where A600 is the absorbance at 600 (nm) and X is the thickness of film (mm).
2.4. Water resistance properties
2.6. Mechanical properties
2.4.1. Moisture content (MC), water solubility (WS) and swelling degree (SD) The film pieces (50 mm × 50 mm) were prepared and weighed (W1), and then dried at 105 °C for 24 h and weighed again (W2). Then the dried samples were immersed into 40 mL distilled water at room temperature for 24 h. Undissolved film portions were filtered out, and the films were weighed after removal of the surface water with blotting paper (W3), then finally dried in an oven at 105 °C until a constant weight (W4) was reached. The MC (%), SD (%), and WS (%) values of the films were determined by the following equations (Zhang et al., 2019):
The tensile strength (TS) and elongation at break (EAB) of the films were determined with a TA.XI Plus Texture Analyzer (Super Technology Instruments Co., Ltd., China) according to the method of ASTM D882-18. Films were cut into strips (80 mm×15 mm) prior to analysis. The test speed was 60 mm/min, and the initial distance was 40 mm.
MC(%) =
W1 − W2 × 100% W1
2.7. Antimicrobial test The antimicrobial activity of the PS/CS films without and with CA against Staphylococcus aureus and Escherichia coli was evaluated by measuring the diameter of the disk inhibition zone. Broth cultures containing 106 CFU/mL of pathogens were prepared and spread onto culture medium. Film discs (14.2 mm in diameter) were placed onto the surface of agar plates containing respective culture medium. Then the prepared discs were incubated at 37 °C for 24 h, and the inhibition zones (diameter) were measured with a caliper and recorded in millimeters.
(1)
SD(%) =
W3 − W2 × 100% W2
(2)
WS(%) =
W2 − W4 × 100% W2
(3)
2.8. Statistical analysis 2.4.2. Water contact angle Multiple samples were tested and the data were presented as the average values ± standard deviation. The difference between factors and levels was evaluated by the analysis of variance (ANOVA) using SPSS 22.0 statistical analysis system. Duncan's multiple range tests were used to compare the means to identify which groups were significantly different from other groups (p < 0.05).
Water contact angles of the films were measured by an OCA20 contact angle analyzer (Dataphysics, Germany). A drop of deionized water (approximate 4 μL) was deposited onto the surface of the film, and the photo was taken at the same time. 2.4.3. Water vapor permeability (WVP) The WVP of the PS/CS composite films cross-linked without and with CA were measured by a PERME W3/031 WVP Tester (Lab think Instruments Co., Ltd. Jinan, China) according to the standard testing method of ASTM E96-16.
3. Results and discussion 3.1. Structural characterization 3.1.1. FTIR spectra FTIR was carried out to determine the possible functional chemical groups and molecular interactions of the cross-linked biopolymer films (Garavand et al., 2017). Fig. 1a showed the FT-IR spectra of CA and the PS/CS film cross-linked without and with CA. The spectrum of citric acid showed characteristic peaks at 3290 cm−1, 1745 cm−1 and 1698 cm−1, corresponding to O-H stretching and C=O stretching of carboxylic acid, respectively (Sampatrao et al., 2018). For the PS/CS film, the characteristic peak at a wavenumber of 3285 cm−1 was associated with the stretching vibration of the –OH group and the –NH2 group (Zhang et al., 2019). The position of the –OH group was almost consistent with the finding of Yusof, Shukur, Illias, and Kadir (2014) that appears at 3288 cm−1 with the blending of starch and chitosan. The absorption bands at 2926 cm−1 and 1595 cm−1 corresponded to CH stretching and N–H bending, respectively (Zhong et al., 2011). After adding CA into PS/CS film, a new peak appeared at 1724 cm−1, which could be attributed to the esterification between citric acid and either starch or chitosan, demonstrating the formation of chemical linkages among them (Reddy & Yang, 2010; Seligra et al., 2016; Shi et al., 2008). In addition, the peak at 1724 cm−1 intensified with increasing CA concentration, indicating the increased crosslinking
2.4.4. Moisture sorption Moisture sorption of the PS/CS/CA composite films was measured according to Gomaa's method with some modification (Gomaa, Hifney, Fawzy, & Gawad, 2018). The films were cut to 40 mm×40 mm strips, dried to a constant weight at 80 °C and placed in a desiccator maintained at 75% RH and 25 °C. The changes in the weight of the films were determined at fixed time intervals and the moisture sorption of the films was calculated as follows:
MS(%) =
Wt − W0 × 100% W0
(4)
Where Wo and Wt were the initial dry weight and the weight of the film sample at regular time, respectively. 2.5. Film thickness, density and opacity The film thickness was determined by taking ten measurements at different positions with a digital micrometer (Zhongtian Experimental Instrument Co. Ltd., Zhengzhou, China). Film density was calculated as the ratio of film weight to its volume (calculated according to the area 3
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Fig. 2. X-ray diffraction patterns of PS/CS films, PS/CS/CA 5% films, PS/CS/CA 15% films, and PS/CS/CA 20% films.
the composite films were obtained by X-ray diffraction (XRD) analysis. Fig. 2 showed XRD patterns of the PS/CS film cross-linked without and with CA. Characteristic peaks of PS/CS film at around 2θ = 17.14°, 19.76° and 22.14° could be attributed to the typical B-type crystals of starch and the regular crystal lattice of chitosan, which were in agreement with previous publications (Mathew, Brahmakumar, & Abraham, 2006; Ren et al., 2017; Suriyatem et al., 2018). When CA were incorporated into these two components, the intensity of characteristic XRD peaks decreased and some even disappeared, and particularly in the cross-linked films at 20% CA concentration, almost one single peak displayed at 17.55°, demonstrating that the intermolecular interactions between PS and CS limited the molecular chain segment movements, restrained their crystallization process and thus reduced the crystallinity of the matrix (Mathew et al., 2006; Ren et al., 2017; Shi et al., 2008; Zhong et al., 2011). The present result was in accordance with Reddy and Yang (2010) and Nataraj, Sakkara, Meghwal, and Reddy (2018), where the crystallinity of starch film and chitosan film decreased with the addition of CA, attributing to the crosslinking reaction caused by CA.
Fig. 1. FTIR spectra of CA and PS/CS films without and with CA (a) and the schematic illustration of possible structure of cross-linked system among CA, PS, and CS (b).
reaction. As indicated in Fig. 1b, the molecular interaction in the filmforming components was further illustrated in the form of a schematic diagram based on the results of the FT-IR spectra. Furthermore, the broad band around 3285 cm−1 became less intense when CA content increased to 15%, indicating the free O-H decreased due to the crosslinking interactions (esterification) (Li, Chen, Li, Bai, & Li, 2012; Seligra et al., 2016; Sun et al., 2017; Liu et al., 2017), as illustrated in Fig. 1b. However, this peak strengthened again for the films with 20% CA addition (Fig. 1a), probably due to excessive CA that unable to crosslink with the biopolymer matrix would cause an increase in the number of the hydroxyl and carboxyl groups and, thus, of the hydroxyl peak height (Shi et al., 2008). Similar observations were reported by Sun et al.(2014) in Chitosan films incorporated with gallic acid and Lei et al. (2019) in pectin-konjac glucomannan composite films incorporated with tea polyphenol, respectively, which indicated that the existence of excessive gallic acid or tea polyphenol would strengthen the peak around 3300 cm−1 (due to OH stretching). The results of ATR-FTIR suggested that crosslinking interactions occurred, which may subsequently affect the properties of PS/CS films.
3.1.3. Film morphology The microstructure and biopolymer compatibility of the developed films were analyzed by SEM, and micrographs of surface and crosssection of the films were shown in Fig. 3. Micrographs of the PS/CS film revealed smooth surfaces and compact structures without any noticeable pores or cracks, indicating the high compatibility and good filmforming properties of the two substances, regardless of whether crosslinking or not. This agreed with the findings of Bonilla et al. (2013) and Ren et al. (2017), who found a good molecular miscibility between starch and chitosan. However, it should be noted that, while the uncross-linked PS/CS film was relatively smooth, the films cross-linked with citric acid presented textured surfaces, particularly when a high concentration of CA (20%) was loaded, which might affect their physical properties. The starch-chitosan blend films with more compact structure and coarser surface due to the networking introduced by ferulic acid has also been reported by Mathew and Abraham (2008). On the other hand, the excessive citric acid crystals (marked by the red circles in Fig. 3) might solidify on sample with 20% CA, promoting a coarser surface on PS/CS films (Azeredo et al., 2015). Similarly, Liu et al. (2017) also observed large crystals on the surface of chitosan films when incorporating 1.5% or 2.0% of protocatechuic acid. VHX-1000C microscopic system was further employed to observe the surface roughness of the films and the resultant 3D surface
3.1.2. XRD The influence of crosslinking agents on the crystalline structure of 4
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Fig. 3. SEM micrographs of PS/CS films, PS/CS/CA 5% films, PS/CS/CA 15% films, and PS/CS/CA 20% films.
Fig. 4. 3D surface topography images of PS/CS films, PS/CS/CA 5% films, PS/CS/CA 15% films, and PS/CS/CA 20% films.
during the first heating cycle, in which all films showed one single endothermic peak. And as shown in Fig. 5b, the peak disappeared after the second heating cycle of the same sample, corresponding to water evaporation (Mathew et al., 2006). The maximum of this water evaporation peak became weaken and shifted from 82.3 to 122.1 °C when CA was incorporated into the PS/CS films, indicating the interactions of cross-linkages would hinder water evaporation (Mathew et al., 2006; Uranga et al., 2019). In order to further explore the thermal stability of the PS/CS/CA composite films, thermogravimetric analysis was utilized to analyze the structural interaction between polymers. The thermal degradation of CA and PS/CS films without and with CA as a function of temperature could be observed in Fig. 6. The weight loss at 40–150 °C could be attributed to the evaporation of weakly physically and strongly chemically bound water for all samples, as also shown by DSC in Fig. 5. It is
topography images were shown in Fig. 4. It can be observed from the 3D images that while the surface topographies of PS/CS blend films were relatively smooth and flat, the CA cross-linked PS/CS films had more protrusions than the uncross-linked films especially at high CA concentration, indicating the increased surface roughness (Gong et al., 2015; Xiong et al., 2014). The 3D surface topography image of PS/CS/ CA composite film was somewhat in agreement with those of ferulic acid incorporated starch-chitosan blend films studied by atomic force microscopy (Mathew & Abraham, 2008), which also exhibited a heterogenous surface with small patches of particles introduced by ferulic acid crosslinking. 3.1.4. Thermal analysis The DSC thermograms of PS/CS films cross-linked without and with CA were presented in Fig. 5. Fig. 5a is the DSC thermogram of the films 5
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Fig. 5. DSC thermograms of PS/CS films, PS/CS/CA 5% films, PS/CS/CA 15% films, and PS/CS/CA 20% films during the first (a) and second (b) heating cycles.
with 15% CA compared to the control, which could be indicative of stronger intermolecular interactions among the components (Li et al., 2019; Mathew et al., 2006), as previously confirmed by FTIR results. Similar results have also been found in starch-based film cross-linked with citric acid (Reddy & Yang, 2010). The structural characterization of PS/CS films preliminarily indicated the presence of crosslinking effect with CA addition, which could further enhance the interactions between molecules. These were further demonstrated by the water resistance, physical and mechanical properties of the films as given below.
worthwhile to mention that similar TG curve of CA in Fig. 6a has been reported by other authors (Thomas, Arun, Remya, & Nair, 2009). The major peak appeared around 212 °C was due to the decomposition of CA (Uranga et al., 2019). The thermograms of the composite films also showed a second stage of weight loss in the temperature range 200–400 °C and was caused by thermal degradation of the films, corresponding to complex processes including the dehydration of the saccharide rings, depolymerization and decomposition of the acetylated and deacetylated units of polymer (Mathew & Abraham, 2008; Mathew et al., 2006). No significant difference in the thermal stability of the control and citric acid incorporated films could be observed from TGA curves in Fig. 5. The stages of weight loss can be investigated better in the DTG curves. The maximum temperature corresponding to each stage seemed to appear at higher temperatures when CA was incorporated into the films, increasing by ∼10 °C and 5 °C for the films
3.2. Water resistance properties 3.2.1. Moisture content, water solubility and swelling degree Moisture content was determined to evaluate the amount of
Fig. 6. TGA and DTG curves of CA, PS/CS films, PS/CS/CA 5% films, and PS/CS/CA 15% films. 6
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Table 1 Moisture content (MC), water solubility (WS), swelling degree (SD) and water contact angle (WCA) of PS/CS films without and with CA. CA
MC
WS
SD
WCA
(w/w)
(%)
(%)
(%)
(°)
0%
17.91 ± 1.11a
50.65 ± 2.15a
686.4 ± 39.6a
5%
17.47 ± 0.29 ab
28.63 ± 1.34b
346.8 ± 26.1b
10%
16.13 ± 0.77bc
24.35 ± 0.66c
184.9 ± 3.1c
15%
16.01 ± 1.20bc
21.60 ± 1.00d
98.1 ± 8.8e
20%
11.14 ± 0.44d
26.41 ± 0.84bc
143.3 ± 4.6d
Values are presented as mean ± standard deviation. Different letters in the same column indicate significant differences (p < 0.05).
moisture in the film. As can be seen from Table 1, the moisture content of the PS/CS films gradually decreased as the increasing of CA content, ranging from 11.14% to 17.91%, in agreement with the weight loss associated to the first step of TGA curves (Fig. 6). Some other studies reported similar findings of films cross-linked with CA (Olsson et al., 2013; Seligra et al., 2016). The decrease of MC in PS/CS/CA films supported the proposal about crosslinking and the consequent decrease of free hydrophilic groups in the films, as previously illustrated in Fig. 1. Water solubility of PS/CS films cross-linked without and with CA was presented in Table 1. The results of WS, decreasing from 50.65 ± 0.02% for control PS/CS films to 21.60 ± 0.08% for PS/CS/ CA 15% films, indicated that the films were less soluble by adding CA. This may be due to the decreased availability of hydrophilic hydroxyl groups and generated hydrophobic ester groups between citric acid and the polysaccharides, resulting in a denser structure and corresponding improved water insolubility of PS/CS/CA compared to films without CA (Reddy & Yang, 2010; Garavand et al., 2017). However, the WS of PS/ CS/CA films increased again when added 20% CA, probably ascribed to the extra-added CA that unable to be cross-linked with polymers, thus leading to more hydroxyl groups and attracting water (Ghanbarzadeh, Almasi, & Entezami, 2011). Swelling degree of PS/CS films cross-linked without and with CA was also presented in Table 1. Cross-linking can cause the films to become more insoluble due to chemical interactions between the chitosan and starch molecules (Li et al., 2013). It could be observed that the SD of the PS/CS/CA films was a significantly lower than that of the PS/CS films (p < 0.05), indicating a lower free volume between knots and a higher crosslinking interactions between PS and CS, which reduced the number of hydrogen bonds to water and created a more dense structure with better resistance (Reddy & Yang, 2010; Suriyatem et al., 2018). In particular, SD of the PS/CS/CA 15% films significantly decreased from 686.4% to 98.1%.
3.2.2. Water contact angle Water contact angle (WCA) was determined to evaluate the hydrophobic nature of the films as well as wettability of their surface. Table 1 showed the WCA values of the PS/CS films cross-linked without and with CA. The WCA of the PS/CS films increased from 34.47° to 56.43° (with addition of 15% CA), indicating that the surface hydrophobicity of PS/CS films was markedly improved. This could be related to the formation of hydrophobic ester groups between citric acid and the polysaccharides, leading to a decrease in the number of polar groups as shown by FTIR analysis (Fig. 1), thus hindering the water absorption from the film surface (Uranga et al., 2019). Moreover, it has been reported that the rough surfaces may have substantially larger contact angles due to heterogeneous wetting (Xiong et al., 2014). The increased surface roughness of CA crosslinked films, evident in the SEM and 3D surface topography images (Figs. 3 and 4), could probably result in larger WCA values. However, WCA value decreased to 51.73° when incorporated with 20% CA, possibly due to the free hydrophilic groups (-COOH and -OH) from excessive addition of CA which solidified crystals on the films’ surface (as confirmed by FTIR spectra in Fig. 1 and shown in Fig. 3). Similar changes of WCA in polysaccharide-based films carrying tea polyphenol were also observed by other researchers (Feng et al., 2018; Lei et al., 2019). 3.2.3. Water vapor permeability Fig. 7a showed the WVP values of PS/CS films cross-linked without and with CA. The WVP of the PS/CS film (3.03 ×10−12 g cm/cm2·s·Pa) was close to the values for corn starch/chitosan films in previous reports (1.22×10−12–3.04×10−12 g cm/cm2·s·Pa) by Ren et al. (2017). The WVP of the CA cross-linked PS/CS films was significantly lower than the WVP of the PS/CS film (p < 0.05), ranging from 2.05 ×10−12 g cm/cm2·s·Pa to 2.81 ×10−12 g cm/cm2·s·Pa. It had been reported that the WVP of a film depended on the number of available polar groups that the polymer contains. The decreased WVP values when added CA might be ascribed to the replacement of hydrophilic hydroxyl groups with hydrophobic ester groups (Garavand et al., 2017). 7
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Fig. 7. Water vapor permeability (a) and moisture sorption curve (b) of PS/CS films without and with CA.
amount of solid content in film-forming solution and thus increased the film thickness. Moreover, with the increase in CA concentration, the crosslinking interactions would cause tighter binding between polymers, resulting in more compact film structure and thereby increasing the density of the PS/CS/CA films (Sun et al., 2017; Wu et al.,. 2019). Table 2 also showed the opacity values as a function of CA concentration on the PS/CS films. Generally, the greater the opacity value, the less transparent were the films. It could be observed that the PS/CS/CA films were less transparent (with higher opacity value) than those of PS/CS films. A former study reported by Li et al. (2019) corroborated the above-mentioned changes in opacity, ascribed to the reaction between crosslinker (CA) and matrix, resulting in a tougher surface (evident in Figs. 3 and 4) and corresponding higher opacity. Nevertheless, it is noteworthy that the composite films still showed good light transmittance that could favor consumer to see the products when used as packaging materials.
On the other hand, the cross-linking effect after CA addition, led to a denser film structure with strong interfacial interaction, which was also beneficial to improve water barrier ability (Garavand et al., 2017; Lei et al., 2019). Seligra et al. (2016) also reported a decrease in the WVP values of starch–glycerol films, when adding CA as crosslinking agent. However, the WVP of PS/CS/CA films increased when added 20% CA. This could be probably explained by the plasticizing role of excessive CA addition, which could increase the mobility of the inter-chain and chain spaces, leading to increasing film diffusivity and high speed of water vapor transmission (Garavand et al., 2017; Ghanbarzadeh et al., 2011). Similar changing tendency in WVP have been reported by Mathew and Abraham (2008) for ferulic acid incorporated starchchitosan blend films and Ghanbarzadeh et al. (2011) for citric acid incorporated starch films. 3.2.4. Moisture sorption The results of the moisture sorption as a function of time of the PS/ CS films without and with CA were depicted in Fig. 7b. It could be observed that the moisture sorption of PS/CS and PS/CS/CA composite films increased with increasing time and reached balance at the 40th hour. And the moisture sorption rate at the initial stages was more rapid and declined with increasing time. Similar results were also obtained by Gomaa, Hifney, Fawzy, and Abdel-Gawad (2018). Moisture sorption of the PS/CS composite films decreased with the increase of CA content, reaching a minimal MS equilibrium value of ∼10.1% for films with 15% CA, and increased again when 20% CA was incorporated. As mentioned above, the supposed reason can be explained by the fact that cross-linking effect of CA resulted in hydrophobic films with denser structure, and thereby improved their water resistance. Whereas, the presence of numerous hydrophilic hydroxyl groups from extra-added CA might tend to interact with water molecules (Garavand et al., 2017), as evidenced by the results of FTIR and WCA.
3.3.2. Mechanical properties The effects of different concentrations of CA on the mechanical properties of PS/CS films were presented in Table 2. The presence of CA caused significant (p < 0.05) differences in the TS and EAB. PS/CS films had relatively lower mechanical properties, TS and EAB of which were 9.72 ± 0.87 MPa and 5.70 ± 0.01%, respectively. Generally, it had been reported that the incorporation of increasing amount of crosslinkers led to improved TS and decreased EAB of biopolymer films (Garavand et al., 2017). As seen from Table 2, TS of the PS/CS composite films increased with increasing of CA concentration, and reached a maximum (29% higher than uncross-linked film) at 15% CA concentration, then slightly declined at 20% CA concentration. While EAB increased with increasing of CA concentration. As seen from Table 1, since the moisture content of the CA crosslinked PS/CS films was lower than the uncrosslinked films, the change in mechanical properties can not be attributed to the plasticizing effect of water. The improved TS obtained by the addition of CA could be due to intra and inter-molecular crosslinking among PS, CS, and CA (as illustrated in Fig. 1b). This interaction contributed to a more compact structure and thereby improved TS of the PS/CS composite films (Mathew & Abraham, 2008). On the other hand, CA may act both as a crosslinking agent and a plasticizer in the biopolymer films, especially at high contents. The extra (not-reacted) portion of citric acid probably acts as a plasticizer and reduces the interactions between the macromolecules, with consequent TS decrement and EAB increase (Garavand et al., 2017; Wang, Ren, Li, Sun, & Liu, 2014). Comparable results were reported by Mathew & Abraham, (2008) and Wang et al. (2014). Wang
3.3. Physical and mechanical properties 3.3.1. Thickness, density and opacity As seen from Table 2, the incorporation of CA promoted an increase in film thickness and density in comparison with the control PS/CS film, and this augmentation was more pronounced in the films with higher CA content (p < 0.05). The thicknesses of the films is directly proportional to the concentration of solids in the formulation, and can be controlled by weighing samples of the blended solution to the same mass prior to casting onto the 250 mm × 500 mm glass plate (Li et al., 2013). The presence of citric acid would have contributed to the more 8
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Table 2 Physical and mechanical properties of PS/CS films without and with CA. CA (w/w) 0% 5% 10% 15% 20%
Thickness (μm) 76.67 80.00 81.33 82.67 86.00
Density
Opacity
−3
± ± ± ± ±
1.53c 1.00b 2.08b 1.53b 1.73a
(g·cm 1.243 1.248 1.251 1.452 1.663
) ± ± ± ± ±
−1
(mm ) 0.92 ± 0.11c 0.98 ± 0.02c 1.04 ± 0.09bc 1.34 ± 0.02b 1.98 ± 0.10a
0.02c 0.01c 0.03c 0.02b 0.05a
TS
EAB
(MPa) 9.72 ± 0.87b 9.27 ± 0.09b 12.53 ± 0.84a 12.55 ± 0.97a 10.49 ± 0.38b
(%) 5.70 6.39 7.13 7.49 8.96
± ± ± ± ±
0.1d 0.19c 0.01b 0.41b 0.62a
Values are presented as mean ± standard deviation. Different letters in the same column indicate significant differences (p < 0.05).
Conflict of interest statement
Table 3 Antimicrobial properties of PS/CS films without and with CA. CA
Inhibition zone diameter (mm)
(w/w) 0% 5% 10% 15% 20%
E. coli (-) 16.48 ± 0.13c 17.09 ± 0.41b 17.14 ± 0.23b 17.32 ± 0.39b 18.14 ± 0.12a
The authors declare no conflict of interest in the publication of this article. S. aureus (+) 16.32 ± 0.33c 17.17 ± 0.64bc 17.99 ± 1.45 ab 18.23 ± 0.70 ab 18.93 ± 0.62a
Acknowledgments This work was supported by Seedling Project of Sichuan Science and Technology Department (2018126), General Program of Natural Science of Sichuan Education Department (16ZB0057).
Values are presented as mean ± standard deviation. Different letters in the same column indicate significant differences (p < 0.05).
Appendix A. Supplementary data
et al. (2014) investigated the effect of citric acid on the mechanical behavior of polyvinyl alcohol/xylan composite films. And they observed that films with 10 wt% citric acid increased TS, when compared to the control film, evidencing a crosslinking effect. However, citric acid concentrations higher than 10 wt% caused the TS to decrease and the EAB to increase, suggesting a plasticizing effect. On the basis of all the collected data, it can be concluded that incorporation of CA as a crosslinking agent in the desired range can modify the properties of PS/CS films, otherwise the excess amount acts as a plasticizer and has an unfavorable impact.
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3.4. Antimicrobial properties The inhibitory effects of PS/CS/CA films on the growth of the two bacteria including the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli bacteria were summarized in Table 3. As could be observed from Table 3, the control PS/CS films showed some inhibitory effect on Escherichia coli and Staphylococcus aureus, which was due to the antimicrobial nature of chitosan (Nataraj et al., 2018). And the diameter of inhibition zones increased as the concentration of CA increased, which was ascribed to the fact that CA has inhibitory effects against some bacteria (Mahmoud, 2014). The PS/CS/CA films with antibacterial properties indicated that they would be beneficial for its practical application in the future. 3. Conclusions PS and CS blend films cross-linked without and with CA were developed and their structural, physical and antimicrobial properties were characterized. The FTIR, XRD and DSC analysis indicated the CA crosslinking interactions occurred between PS and CS. The PS/CS/CA films exhibited a homogenous and denser structure with higher surface roughness as well as enhanced water resistance, mechanical and antimicrobial properties compared with uncross-linked films. Particularly, the results indicated that the films cross-linked with 15% CA showed the best comprehensive properties among all films. However, overloading of CA (20%) would slightly decrease mechanical and water resistance properties, possibly due to the plasticizing role of CA. This study provided the appropriate formula of developing CA crosslinked PS/CS films with desirable properties that possess potential in food preservation and packaging industry. 9
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