Characterization of chitosan based polyelectrolyte films incorporated with OSA-modified gum arabic-stabilized cinnamon essential oil emulsions

International Journal of Biological Macromolecules 150 (2020) 362–370

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Characterization of chitosan based polyelectrolyte films incorporated with OSA-modified gum arabic-stabilized cinnamon essential oil emulsions Tian Xu a, ChengCheng Gao a, Xiao Feng a, Di Wu a, Linghan Meng a, Weiwei Cheng a, Yan Zhang b, Xiaozhi Tang a,⁎ a College of Food Science and Engineering/Collaborative Innovation Center for Modern Grain Circulation and Safety/Key Laboratory of Grains and Oils Quality Control and Processing, Nanjing University of Finance and Economics, Nanjing 210023, China b Hebei Key Laboratory of Food Safety, Hebei Food Inspection and Research Institute, Shijiazhuang 050091, China

a r t i c l e

i n f o

Article history: Received 16 December 2019 Received in revised form 3 February 2020 Accepted 10 February 2020 Available online 11 February 2020 Keywords: Chitosan films Gum arabic Octenyl succinic anhydride Cinnamon essential oil Emulsion

a b s t r a c t Octenyl succinic anhydride (OSA) modified gum arabic (GA) was synthesized and used as an emulsifier to stabilize cinnamon essential oil (CEO) emulsions. The structure and properties of chitosan based polyelectrolyte films incorporated with above OSA-GA stabilized CEO emulsions were investigated. Results showed that OSA modification introduced the hydrophobic groups, which greatly influenced the emulsification capability of GA. The antimicrobial activities of CEO emulsions were significantly enhanced by the synergistic effect of GA modification and ultrasonic treatment. When the proportions of CEO emulsion increased, the improved water barrier properties but deteriorated tensile properties of films were observed. The retention of CEO during storage was prolonged to 20 days and the release of CEO in food simulant was effectively inhibited as emulsion ratios increased to 20%, indicating the effectiveness of the system for CEO delivery. As a result, the antimicrobial activities of films significantly enhanced with the innovative incorporation of OSA-GA stabilized CEO emulsions. © 2020 Elsevier B.V. All rights reserved.

1. Introduction Traditional plastic packaging films are not biodegradable, which has caused great harm to the environment. The possible migration of chemicals in these films to food also poses a potential threat to the human body. With the great environmental concerns and the consumer demand for safe food products, development of biodegradable and edible packaging films becomes the research trend [1]. Furthermore, edible films may operate as carriers for natural antioxidant and antimicrobial agents like essential oils. Such active and edible films have shown great potentials in extending the shelf life of different types of food [2,3]. Essential oils (EOs) are oily liquids extracted from different parts of aromatic plants [4]. Most EOs have shown strong antioxidant and antimicrobial capacity [5]. Among all EOs, cinnamon essential oil (CEO) has attracted more attention due to its superior antimicrobial activities and wide applications for food preservation [6,7]. Cinnamaldehyde, the major chemical component of CEO (80.01%), has been identified as the main reason of its antimicrobial properties [6]. However, most EOs including CEO easily volatilize at room temperature and have low water solubility, which might lead to the increased risks of oiling off and

⁎ Corresponding author at: College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210023, China. E-mail address: [email protected] (X. Tang).

https://doi.org/10.1016/j.ijbiomac.2020.02.108 0141-8130/© 2020 Elsevier B.V. All rights reserved.

poor dispersion within hydrophilic polysaccharide film matrix [8]. To overcome this, EOs were often stabilized by surfactants and different biopolymers to increase the encapsulation efficiency and compatibility of EOs in films and improve their antimicrobial activities [9–11]. Gum Arabic (GA) is an anionic polyelectrolyte heteropolysaccharide extracted from the branches and trunks of Acacia trees [12]. Generally, it can be divided into three fractions including arabino galactan (Mw, ~300 KDa), arabino galactan protein (Mw, ~1500 KDa), and glyco protein (Mw, ~250 kDa), which represents about 88%, 10% and below 2% of the total gum, respectively [13]. The emulsifying property is one of the most important functional properties of GA. The emulsifying effect of GA on oil-in-water emulsion systems were mainly resulted from changing the apparent viscosities of food system or adsorbing at oil-water interface to produce a viscoelastic film, thereby reducing the surface tension and stabilizing emulsions [14]. It has been reported that GA improved the dispersibility and retention of the EOs in emulsions/coatings, and greatly enhanced their antimicrobial activities [15,16]. However, the emulsification capacity of GA is limited and higher concentrations of GA are usually required to obtain stable oil-in-water emulsions due to the small proportion of emulsification components in structure [13]. Octenyl succinic anhydride (OSA), an excellent esterification modifier, was reported to be grafted onto GA molecules to enhance the emulsifying performance of GA [13,17]. The European Food Safety Agency has confirmed the safety of OSA-GA and authorized the use of OSA-GA as food additives [18].

T. Xu et al. / International Journal of Biological Macromolecules 150 (2020) 362–370

In our previous work, polyelectrolyte films based on chitosan (CS), GA and CEO were developed through one-step blending [19,20]. In the system, GA was mainly acted as a film-forming polymer and entangled with CS molecules through electrostatic interactions. CEO was caught by GA and embedded in the core of CS-GA intertwined structure, leading to the high stability and retention of CEO in film matrix. However, high proportions of GA were required (CS:GA = 1:2) to obtain satisfactory retention and release properties since GA was needed to provide more chances for CS chains to be connected through electrostatic interactions and enough emulsification capacity to catch CEO [19]. As a result, mechanical properties especially the tensile strength were significantly impaired, which decreased about 73% [20]. In this study, OSA modified GA was applied as an emulsifier/stabilizer to stabilize CEO emulsions, and then the CEO emulsions were incorporated into CS film matrix. The synergistic effect of GA modification and ultrasonic treatment on stability and antibacterial properties of CEO emulsions was studied. Further, the effects of different ratios of OSA-GA stabilized CEO emulsions on the structure, physical and antimicrobial properties of chitosan-based active films were investigated. The hypothesis would be the resultant polyelectrolyte emulsion films further improved the CEO retention and antimicrobial properties of films, as well as reduced the deterioration of mechanical strength. 2. Materials and methods 2.1. Materials Gum Arabic (GA, Acacia Senegal, density = 1.35 g/mL, Mw = 3.0 × 105 g/moL) was purchased from Macklin Biotech Co., Ltd. (Shanghai, China). Octenyl succinate anhydride (OSA, 99% purity, viscosity ≤ 45 cP) was supplied by Sliken Trade Co., Ltd. (Shenzhen, China). Cinnamon essential oil (CEO, 85% purity) was obtained from Dongshi Flavor Co., Ltd. (Shanghai, China). Chitosan with degree of deacetylation of 90.27% and molecular weight of 5.0 × 104 g/moL was provided by Jinan Haidebei Ocean Biological Engineering Co., Ltd. (Shandong, China). All other chemicals were used with analytical grade. 2.2. Preparation of OSA-GA OSA-GA was prepared by the method of Li et al. [14] with some modifications. GA solution (25%, w/v) was obtained through dissolving GA in deionized water, followed by high-speed stirring. 3% of OSA (g/g GA) was first diluted with ethanol (0.25% v/v) and then added drop wise into GA solution at 40 °C for 1.5 h. During the reaction, the pH was maintained at 8.0 by NaOH solution (0.5 M) to ensure the positive direction of reaction. The termination was accomplished by adjusting the pH to 6 by HCl solution (0.1 M). The mixture was successively dialyzed by ethanol and water, and finally freeze-dried. OSA-GA powers were obtained after pulverizing and sieving (200 mesh). 2.3. Characterization of OSA-GA 2.3.1. Fourier transform infrared spectroscopy (FT-IR) The structural changes of OSA-GA and the resulted films were determined by a FT-IR spectrometer (Bruker Tensor 27, Germany). Samples were ground into powders with KBr (1:100–1:200). FT-IR spectrums were scanned from 4000 to 500 cm−1. 2.3.2. The degree of substitution (DS) and molecular weight (Mw) The degree of substitution (DS) of OSA-GA was determined by the method previously described [17]. OSA-GA powders were dispersed in NaOH aqueous solution (0.25 M) with magnetic stirring for 30 min. The excessive NaOH was then titrated by HCl with phenolphthalein as indicator. Pure GA was used as blank for simultaneous titration.

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The DS of OSA-GA was then calculated according to following equations: W ð%Þ ¼

DS ¼


V blank −V sample  M  N  100% msample ðgÞ  1000

162  W 100  M−ðM−1Þ  W

ð1Þ

ð2Þ

where, W(%) is% OS substitution in OSA-GA; Vblank and Vsample respectively represent the volume of HCl required for blank and sample titration (mL); M is the molecular weight of OSA (210 g/mol); N is the normality of HCl and 162 is the molecular weight of the glucose unit. The molecular weight (Mw) of GA and OSA-GA was obtained by Gel Permeation Chromatograph (GPC). The measurement was performed under following parameters: E2695 column (in series); column temperature 30 °C; injection volume 50 μL; sodium acetate buffer as mobile phase (0.2 M). 2.3.3. pH value and rheological properties of aqueous solutions GA and OSA-GA samples were dispersed in deionized water to prepare a 10% (w/v) aqueous solution. The pH value of the aqueous solutions was measured by a pH meter (PHS-3C, Jingke Equipment, China). The rheological behavior of the aqueous solutions was performed by a MCR 302 rheometer (AntonPaar, Austria) with plate-plate geometry (50 mm). The flow behavior index (n) and the consistency index (K) were obtained, which closely fitted the Ostwald de Waale model (R2 N 0.99) at the shear rate range of 1–300 s−1. 2.4. Preparation of CEO emulsions with GA or OSA-GA as an emulsifier GA and OSA-GA samples were separately dispersed in deionized water (10% w/v) and then cinnamon essential oil (CEO) was slowly added (the mass ratio of GA/OSA-GA and CEO was 8:1) to prepared CEO emulsions. Four types of CEO emulsions were prepared: GA/CEO emulsions through regular homogenization (GA/CEO); GA/CEO emulsions through ultrasonic treatment (GA/CEO-U); OSA-GA/CEO emulsions through regular homogenization (OSA-GA/CEO) and OSA-GA/ CEO emulsions through ultrasonic treatment (OSA-GA/CEO-U). The parameters for high-speed homogenization and ultrasonic treatment were 17,000 rpm, 4 min (IKA T18-DigitalUltra-Turrax, Staufen, Germany) and 375 W, 8 min (SCIENTZ-1500F, Ningbo Scientz, Ningbo, China) respectively. 2.5. Emulsion stability Four types of CEO emulsions were stored at 4 °C for 1 week. The particle size and polydispersity index (PDI) of emulsions were measured with a Nano-ZS90 Zetasizer (Malvern, UK) during storage. 2.6. Antimicrobial assay of emulsions 2.6.1. Bacterial strains The representative bacteria including Staphylococcus aureus (ATCC 25923, S. aureus) and Eschericha coli (ATCC 25922, E. coli) were provided by Key Laboratory of Grains and Oils Quality Control and Processing of Nanjing University of Finance and Economics (Nanjing, China). Bacterial strains were incubated in nutrient broth at 37 °C for 24 h before use. 2.6.2. Zone inhibition test The antimicrobial activities of four types of CEO emulsions were assessed according to a previous study with some modifications [21]. Briefly, the sterilized filter discs (ø = 6 mm) were impregnated in emulsions for 6 h, and then taken out for natural drying. Dried discs were placed on the medium surface which had been previously seeded with bacterial suspension (150 μL) at approximate 106 CFU/mL, then dripped

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with 10 μL of the corresponding emulsions respectively. All plates were incubated at 37 °C for 24 h and the antimicrobial activities of CEO emulsions were evaluated by observing the diameter of inhibition zone.

fixed with the initial gap of 40 mm, and stretched at the speed of 25 mm/min. For each sample, results were obtained from at least 8 replicates.

2.6.3. MIC and MBC determination The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated according to the method previously described [22]. Pure CEO and a group of CEO emulsions with the best antimicrobial capacity were separately mixed with nutrient broth to achieve a concentration of 5 mg/mL. Concentration series were prepared with a serial two fold dilution method, then the bacterial suspension (200 μL) with a concentration of 106 CFU/mL was added to the tubes. The mixtures were transferred to incubate at 37 °C for 24 h and the optical density (OD) at 650 nm was recorded with a SpectraMax–M2e microplate reader (Molecular devices, China). The lowest concentration that manifested no visible growth (OD change ≤0.05) after incubation was reported as the MIC. MBC was recorded as the lowest concentration for aseptic growth after transferring those with the specific concentration (≥ MIC) to agar medium and incubating at 37oC for 24 h. Tests were performed in three replicates.

2.8.4. X-ray diffraction (XRD) X-ray diffraction (XRD) patterns of chitosan, gum arabic powders and chitosan-based films were obtained using an X-ray diffractometer (D/max-2500/PC, Rigaku, Japan) in tubes (Cu-Kα radiation, wavelength of 0.154 nm). All scans were taken between 2θ = 5° to 40° with a step speed of 2°/min.

2.7. Preparation of chitosan-based active films Chitosan powders (1.5% w/v) were dissolved in 1% v/v acetic acid solution followed by mixing with glycerol (20% w/w of chitosan). After that, OSA-GA/CEO-U emulsions were added to the above solutions to reach their final concentrations of 5%, 10%, 15% and 20% (v/v chitosan solutions). The above film-forming dispersions were homogenized at 12000 rpm for 4 min using an IKA T18 homogenizer (Staufen, Germany). The dispersions were then poured in square polyethylene dishes (12.5 cm side length) and allowed to dry at 45 °C for 12 h. The obtained films were stored at 25 °C and 50% relative humidity prior to further tests. 2.8. Characterization of films 2.8.1. Appearances and the opacity Appearances of film-forming dispersions and films were obtained by a digital camera. The opacity was estimated as described [23]. Film absorbance at 600 nm was measured by an ultraviolet spectrophotometer (Hitachi U-3900, Japan).

2.8.5. CEO loss during storage The loss of CEO from films during storage was evaluated according to a previous study [26] with slight modifications. Film samples (1.5 g) were put in tubes containing 25 mL hexane and then stirred at 25 °C for 24 h. Subsequently, the mixture was severely centrifuged at 11,400 rpm for 10 min, followed by collecting the supernatant for analysis with the wavelength at 318 nm by an ultraviolet spectrophotometer (Hitachi U-3900, Japan). The amount of CEO was determined by an external calibration curve of free CEO in hexane. Loss of CEO was calculated as wt% based on the theoretical amount of CEO in film-forming dispersions. Each film sample was measured in three replicates. 2.8.6. Release of CEO to food simulant The release of CEO was monitored using 60% glycerol solution as simulant media, corresponding to the liquid food system with the water activity of about 0.6–0.7. Around 0.5 g films were introduced to glycerol solution (30 mL) with gentle agitating, then a specific volume of the mixture was sucked out periodically [18]. The CEO content was quantified by ultraviolet spectrophotometer at 318 nm. All tests were run in triplicate. 2.8.7. Antimicrobial activities of chitosan-based active films Antimicrobial activities of the chitosan-based films were determination [27]. Films (1 g) were transferred to the test tubes containing 20 mL nutrient broth, followed by adding 60 μL of bacteria (108 CFU/mL) and subsequently shaking at 37 °C. During the incubation, suspension aliquots were pipetted and logged at optical density of 650 nm every 1 h with a SpectraMax–M2e microplate reader (Molecular devices, China) to obtain bacterial growth kinetics.

2.8.2. The moisture content and water vapor permeability (WVP) The moisture content of films was measured by drying films at 105 °C until a constant weight was achieved, and calculated as the percentage of weight loss based on the original weight of films. Water vapor permeability (WVP) experiments were performed with the LLY11 permeability cups (Yuanmore, China) based on the standard method of ASTM E96–95 [24]. These cups fixed with film discs (ø = 9 cm) were conditioned in a desiccator (25 °C, 75% relative humidity) and weighed every 2 h for at least 12 h. The water vapor transmission rate (WVTR) and WVP were calculated as:

2.9. Statistical analysis

WVTR ¼ Δm=ðA  tÞ

ð3Þ

WVP ¼ WVTR  d=ΔP

ð4Þ

It had been reported that the esterification reaction occurred between the hydroxyl groups of GA and the carbonyl groups of OSA in weak alkaline environment [13,17]. The esterification can be verified by FT-IR, which was shown in Fig. 1. The broad band appearing at 3409 cm−1 corresponded to the stretching vibration of the O\\H or N\\H bond [13,28]. The peaks at 2932 cm−1 and 1624 cm−1 were assigned to the stretching vibrations of C\\H and N\\H bending vibrations, respectively [28]. Compared with GA, the spectrum of OSA-GA showed a new carbonyl (C_O) peak at 1728 cm−1, suggesting that the carbonyl groups of OSA combined with the hydroxyl groups of GA via ester bonds [13]. In addition, no new peaks appeared for the FT-IR patterns of the OSA-GA, indicating that the modification introduced no other substances but the OSA hydrophobic groups. Predictably, the

where, Δm is the mass loss of cups (g); t is the time (h), and A is the effective film area (m2); d is film thickness (mm), and ΔP is the partial pressure between two surfaces (kPa). Tests were performed in three replicates. 2.8.3. Tensile properties Tensile strength (TS) and elongation at break (E) of films were characterized using an XLW tensile tester (Labthink, China) according to the standard method ASTM D882–02 [25]. Film strips (2.5 cm × 9 cm) were

Analysis of variance (ANOVA) was performed to assess the differences in mean values with SPSS 17.0 (SPSS Inc. Chicago, IL, USA). Duncan's multiple range tests were used to analyze the statistical significance at 0.05 level. 3. Results and discussion 3.1. FT-IR analysis of GA and OSA-GA

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Fig. 1. FT-IR spectra of GA and OSA-GA.

introduction of hydrophobic OSA group could enhance the emulsifying properties of GA.

3.2. Physical properties of GA and OSA-GA The degree of substitution (DS), molecular weight (Mw), pH and Ostwald de Waale rheological parameters of GA and OSA-GA were shown in Table 1. The DS of OSA-GA prepared by 3% OSA was 0.054, which was within the ranges described by previous reports [17]. OSA-GA had higher Mw as to GA, suggesting that OSA groups were grafted onto GA molecules through the reaction [13]. In addition, the increased molecular weight of OSA-GA might also be related to the hydrophobic association between OSA alkyl chains [28]. Compared with GA, the pH value of OSA-GA aqueous solutions was lower. Besides, the OSA-GA aqueous solutions showed a decreased flow behavior index (n) and an increased consistency index (K), indicating that OSA-GA aqueous solutions were more pseudoplastic. It had been reported that the highly branched molecular structure of GA contributed to their lower solution viscosity compared with other polysaccharides with similar molecular weight [29]. The increased viscosity of OSA-GA solutions was related to the formation of multi-molecular clusters caused by the hydrophobic association of molecules through OSA alkyl chains [13,30].

Table 1 The degree of substitution (DS), molecular weight (Mw), pH and Ostwald de Waale rheological parameters of GA and OSA-GA. Sample

DS

Mw(∗105g/moL)

pH

n

K(Pa·s)

GA OSA-GA

– 0.054 ± 0.001

3.03 4.21

7.32 ± 0.03 6.78 ± 0.02

0.996 0.978

0.017 0.029

3.3. Stability of CEO emulsions The particle size and polydispersity index (PDI) of the CEO emulsions during storage were presented in Fig. 2. Fig. 2A showed that the particle size of all emulsions gradually increased during storage time. The CEO droplets showed a tendency to coalescence due to its relatively higher polarity and interfacial tension, leading to the increased emulsion particle size in primary stage. After 3 days of storage, the size growth began to level off, indicating that the newly formed droplets achieved thermodynamic equilibrium [31]. Smaller particle size of emulsions always indicated a better stability [32]. The particle size of the CEO emulsions significantly reduced when OSA-GA was applied. The smaller size of emulsions might be due to the enhanced emulsifying ability of OSA-GA which made it faster to adsorb onto the droplets, resulting in a reduced droplet aggregation [28]. From Fig. 2A, further decrease of particle size was observed when OSA-GA emulsification was combined with ultrasonication, due to the generated disruptive shear force by ultrasound treatment for oil droplet decreasing. PDI was an important parameter to represent size distribution and the homogeneity of droplets. Smaller PDI values often implied a narrower particle size distribution with high droplet uniformity [33]. Fig. 2B indicated that the modification of GA and ultrasonication also significantly decreased the PDI of CEO emulsions. It should be noted that the film casting and drying process lasted about 12 h, and the relatively stable CEO emulsions during these steps ensured the high efficiency of CEO to be incorporated into chitosan film matrix. 3.4. Antimicrobial activities of CEO emulsions Zone inhibition test was applied to qualitatively evaluating the antimicrobial activity of CEO emulsions against E. coli (Fig. 3A) and S. aureus (Fig. 3B). Inhibition zone diameters of CEO emulsions were provided in supplemental material Table S.1. Results showed that the GA/CEO

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Fig. 2. The mean particle size (A) and PDI (B) of CEO emulsions during ambient storage.

Fig. 3. Antimicrobial activity of CEO emulsions against E. coli (A) and S. aureus (B). 1–4 in each image represented respectively GA/CEO, GA/CEO-U, OSA-GA/CEO, OSA-GA/CEO-U.

emulsions showed lower sensitivity to E. coli and S. aureus with smaller inhibition area. In contrast, the modification of GA combined with ultrasonication (OSA-GA/CEO-U) remarkably enhanced the antimicrobial properties of the film. The modification of GA introduced hydrophilic carboxyl groups and hydrophobic alkyl chains, which improved the affinity for water and oil, thereby facilitating the dispersion of CEO droplets. Furthermore, the reduced particle size (Fig. 2A) enhanced the diffusion of CEO and increased the contact with microorganisms due to the increase of surface area, thus improving the antimicrobial efficacy [32]. MIC/MBC tests were performed to quantitatively analyzing the antimicrobial capacity of pure CEO and OSA-GA/CEO-U emulsions. MIC was the lowest concentration of additives to inhibit the growth of microorganisms and MBC was the lowest concentration to kill 99.9% of the microorganisms [34]. From Table 2, the MIC of pure CEO for E. coli and

Table 2 The minimum inhibitory concentration (MIC) or minimum bactericidal concentration (MBC) against E. coli and S. aureus were determined to show the antimicrobial activity of pure CEO and CEO emulsion stabilized with OSA-GA. Samples

CEO OSA-GA/CEO-U

E. coli

S. aureus

MIC (mg/mL)

MBC (mg/mL)

MIC (mg/mL)

MBC (mg/mL)

2.5 0.625

2.5 1.25

1.25 0.625

2.5 0.625

S. aureus was 2.5 and 0.625 mg/mL, respectively, while OSA-GA/CEO-U emulsions reduced the MIC of two types of bacteria by 4 and 2 times, respectively. The MBC of pure CEO for both E. coli and S. aureus was 2.5 mg/mL, whereas the OSA-GA/CEO-U emulsions reduced the MBC of two types of bacteria by 2 and 4 times, respectively. These results confirmed the pronounced inhibition effects of CEO emulsions, which was consistent with the previous studies [35–37]. The lower antimicrobial capacity of the pure CEO was because of the low water solubility of CEO inhibiting its interactions with the microbial cell membrane. Nevertheless, the small size effect of the emulsions might accelerate the diffusion of CEO to the microbial cell membrane through passive transport [31]. To be noted, the decreased MIC and MBC meant reduced use of CEO in the film formulation, thereby alleviating their adverse influences on film properties. 3.5. Physical properties of chitosan-based active films The appearances of film-forming dispersions and edible films could influence the acceptance of consumers. As shown in Fig. 4A, the color of film-forming dispersions changed from pale yellow to milky white with the increased content of emulsions. This phenomenon was mainly related to the stronger polyelectrolyte complexation between CS and OSA-GA and the enhanced light scattering by oil droplets [20]. As to films, smooth surfaces were observed for all the films and the film color turned to be yellowish with the increase of emulsion ratios

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Fig. 4. Visual appearance of film-forming dispersions (A) and films (B) with different ratios of CEO emulsions: a, a1–0% emulsion; b, b1–5% emulsion; c, c1–10% emulsion; d, d1–15% emulsion; e, e1–20% emulsion.

(Fig. 4B), which was also supported by the opacity results presented in Table 3. The reason for this is due to increased oil content in the films. It was also reported that Schiff-base reaction between CS and CEO might also contribute to this color changes [38,39]. Physical properties including the moisture content, WVP and mechanical properties of films affected their performance and integrity during packaging. Table 3 showed that the moisture content and WVP of films decreased significantly (p b .05) with the increasing content of the CEO emulsions. For example, the control films (0% emulsion) showed the highest moisture content (22.5%) and WVP (0.35 g·m−1·KPa−1· h−1·10−3), whereas the moisture content significantly decreased to 13% and the WVP decreased to 0.24 g·m−1·KPa−1·h−1·10−3 as the proportion of CEO emulsions reached 20%. Hydrophobic CEO displayed smaller droplets in films in the presence of OSA-GA, which increased the tortuosity factor for water transport in films [23,40]. In addition, the

electrostatic complexation between CS and OSA-GA resulted in hindering the movement of water molecules and thereby enhancing the water barrier properties [19]. The incorporation of CEO emulsions in films caused the significant decrease in both TS and E (p b .05). Since the modification of GA only introduced the hydrophobic groups, the increased amount of OSA-GA due to the incorporation of CEO emulsions would react with chitosan molecules through electrostatic interactions and formed entangled networkglobular structure as described previously [19]. This structure might deteriorate the tensile properties of CS based films, due to the inhibition of the intramolecular hydrogen bonding by stronger electrostatic interactions between CS and OSA-GA [1,23]. Besides, the weakened plasticizing effect caused by the decreased moisture content in film matrix was also an important factor. However, the tensile strength only decreased from 33.6 MPa to 20.7 MPa when increasing CEO emulsion ratios from 0% to

Table 3 The opacity, moisture content, WVP, and mechanical properties of chitosan-based films. Opacity (%) 0% emulsion 5% emulsion 10% emulsion 15% emulsion 20% emulsion

0.83 0.72 1.52 1.88 2.23

± ± ± ± ±

Moisture content (%) 0.18d 0.21d 0.18c 0.15b 0.09a

22.50 18.90 16.32 13.61 13.03

± ± ± ± ±

0.02a 0.01b 0.01c 0.01d 0.01d

WVP (g·m−1·KPa−1·h−1·10−3) 0.35 0.31 0.28 0.26 0.24

± ± ± ± ±

0.01a 0.02b 0.00c 0.01cd 0.01d

TS (MPa) 33.60 28.42 26.01 22.02 20.70

All values were expressed as means ± standard deviation (n ≥ 3). Different letters in same column indicate significantly differences in means.

± ± ± ± ±

E (%) 2.32a 4.29b 3.82b 4.01c 5.23c

38.03 33.88 32.03 30.37 30.33

± ± ± ± ±

1.90a 5.94b 4.32bc 3.22c 4.51c

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Fig. 5. FT-IR (A) and XRD (B) patterns of chitosan-based active films.

20%, meaning the addition OSA-GA-CEO emulsions into CS matrix would be a proper way to reduce the deterioration of mechanical properties of the films as compared to our previous study [20]. 3.6. FT-IR and XRD analysis for chitosan-based active films FT-IR results of chitosan-based films were presented in Fig. 5A. In pure CS film, the broad band at 3445 cm−1 was the –OH stretching. The peak at about 2917 cm−1 and 1657 cm−1 corresponded to the stretching vibration of \\CH and C_O in amide I, respectively. Similar spectrum for CS film has been previously reported [41]. With the increased ratio of OSA-GA-stabilized CEO emulsions in polyelectrolyte films, the \\CH stretching vibration peak gradually disappeared, and the peak at 1657 cm−1 gradually shifted to the left, implying stronger electrostatic interaction between CS and GA [42]. In addition, some new characteristic bands appeared at 3743 cm−1 and 2368 cm−1– 2320 cm−1, and the peak strength increased with the increased emulsion content, which may be related to other characteristic peaks of CEO [43]. XRD profiles were shown in Fig. 5B. Two main diffraction peaks at 2θ = 12° and 19.9° were observed in pure chitosan powders. However, these two peaks shifted to lower angle (11.19° and 18.01°, respectively) and another two new peaks at around 8.32° and 21°appeared when

casted as films (0% emulsion, CS film), which was possibly because of the formation of chitosan acetate in films. With the incorporation of CEO emulsions, the peaks at 8.32°, 11.19° and 18.01° became weaker and even disappeared, which might be explained by the weakened interactions between acetic acid (or acetate) and OH– groups of chitosan and enhanced interactions between CS and OSA-GA. In addition, pure GA powders have a characteristic peak at about 20.3°. The peaks in composite films gradually shifted to the left when increasing rations of CEO emulsions, which confirmed the occurrence of the interactions between CS and GA. Such interactions got enhanced when the content of OSA-GA increased. 3.7. The retention and release properties of chitosan-based active films The loss of CEO in films during storage was shown in Fig. 6A. The increased ratios of emulsions in films resulted in a significant loss of CEO (p b .05). Fresh films (day 0) incorporated with 5% CEO emulsions had a loss about 39.54%, as compared to a 47.31% loss in films with 20% emulsions. Based on our previous study, CEO was captured by OSA-GA and then encapsulated in the core of the chitosan /OSA-GA intertwined structures to ensure the high stability and retention of CEO in film matrix [19]. The increased ratios of emulsions caused the relatively lower ratio of the polymer matrix to emulsified CEO, which decreased the

Fig. 6. The loss (A) and release properties (B) of CEO in chitosan-based active films.

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Fig. 7. The growth inhibiting activity of chitosan-based films on E. coli (A) and S. aureus (B).

encapsulation efficacy of CEO in films. In addition, the loss of CEO was related to less compactness of the films, when higher amount of CEO droplets was incorporated. However, from Fig. 6A, the retention time was significantly extended to 20 days, indicating the effectiveness of emulsion incorporation in improving the retention of CEO in films. To be noted，the prolonged retention time of CEO would broaden the application of antimicrobial films and directly influence the storage quality of produce packaged. Fig. 6B showed the release behaviors of chitosan-based films in 60% glycerol solution. Overall, an initial rapid release of CEO was displayed and then progressively slowed down until a constant level. After immersing in simulation system, CEO in the shallow matrix of films quickly released, and then the internal CEO diffused to the solution by the concentration gradient until liquid-solid balance [18]. The release of CEO in chitosan-based films significantly decreased with the increasing emulsion ratios (p b .05). Within 60 min, about 80% CEO was released for the films containing 5% CEO emulsions. Nevertheless, about 64% CEO was released for the films containing 20% CEO emulsions and the complete release was reached until 500 min. For the films containing higher content of CEO emulsions, the sustained CEO release was obtained due to the enhanced interactions between CS and OSA-GA as shown in Fig. 5, which promoted the entrapment of CEO within polymeric matrix. Besides, the release of CEO caused a reduced concentration gradient between film matrix and the simulant, leading to a lower release rate from film network [19]. The sustained release of CEO in films suggested the effectiveness of emulsions for CEO delivery, which would be beneficial to the antimicrobial capacity of films. 3.8. Antimicrobial activities of chitosan-based active films The growth kinetics curves of E. coli and S. aureus exposed to chitosan-based films were presented in Fig. 7A and Fig. 7B, respectively. Bacteria strains all grew rapidly within 15 h, and then entered a stable growth period. As expected, the antimicrobial activities of the films incorporated with CEO emulsions were significantly stronger than that of pure chitosan film (0% emulsion), and the addition of CEO emulsions had significant contribution to antimicrobial activities. Numerous studies have reported the enhancement of antimicrobial properties of the films when essential oils were introduced in the form of emulsions [21,38,44], and agreed that the bacteriostatic effect of films depended mainly on the content of essential oils and their release kinetics. The higher emulsion content brought higher concentration of CEO and lower release rate of them in the films (Fig. 6B), thus enhancing the continuous destruction to microorganism. It should be noted that the growth of two microorganisms was almost completely inhibited when the emulsion proportion reached 20%.In addition, different kinetic curves for the two strains were displayed, which was most likely related

to the differences in the antimicrobial mechanism of CEO to E. coli and S. aureus [45]. 4. Conclusion In this study, CEO emulsions stabilized by OSA modified GA (OSAGA) were prepared and were successfully introduced into chitosan films. Results clearly showed the stable emulsions with smaller particle size were obtained, which was related to the enhanced emulsifying capacity of OSA-GA. The strong antimicrobial capacity of CEO emulsions was verified by zone inhibition test and MIC/MBC tests. The incorporation of higher proportions of emulsions (up to 20%) slightly reduced the tensile strength but improved water resistance of the complexed films. The higher retention time and the sustained release of CEO from the films contributed to the improved antimicrobial activities of the films against E. coli and S. aureus. In a word, the incorporation of OSAGA stabilized CEO emulsions into CS films would be a better way to protect CEO in edible films and reduce the deterioration of mechanical strength of the polyelectrolyte complexed films. Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2020.02.108. CRediT authorship contribution statement Tian Xu: Conceptualization, Methodology, Software, Writing - original draft, Writing - review & editing. ChengCheng Gao: Data curation. Xiao Feng: Visualization, Investigation. Di Wu: Formal analysis. Linghan Meng: Software, Validation. Weiwei Cheng: Project administration. Yan Zhang: Supervision. Xiaozhi Tang: Conceptualization, Project administration. Acknowledgements This project was supported by the National Natural Science Foundation of China (Grant no. 31801493) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References [1] Z. Shen, D.P. Kamdem, Development and characterization of biodegradable chitosan films containing two essential oils, Int. J. Biol. Macromol. 74 (2015) 289–296. [2] P.K. Dutta, S. Tripathi, G.K. Mehrotra, J. Dutta, Perspectives for chitosan based antimicrobial films in food applications, Food Chem. 114 (2009) 1173–1182. [3] G. Yuan, X. Chen, D. Li, Chitosan films and coatings containing essential oils: the antioxidant and antimicrobial activity, and application in food systems, Food Res. Int. 89 (1) (2016) 117–128. [4] N. Sadgrove, G. Jones, A contemporary introduction to essential oils: chemistry, bioactivity and prospects for Australian agriculture, Agriculture 5 (1) (2015) 48–102.

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