Effect of molecular weight on the properties of chitosan films prepared using electrostatic spraying technique

Carbohydrate Polymers 212 (2019) 197–205

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Effect of molecular weight on the properties of chitosan films prepared using electrostatic spraying technique

T

Yu Zhonga,b, , Chenjun Zhuanga,b,1, Weiqi Gua, Yanyun Zhaob,c, ⁎

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a

Department of Food Science and Technology, Shanghai Jiao Tong University, 800 Dongchuang Road, Shanghai 200240, China SJTU-OSU Innovation Center for Environmental Sustainability and Food Control, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China c Department of Food Science and Technology, 100 Wiegand Hall, Oregon State University, Corvallis, OR 97331-6702, USA b

ARTICLE INFO

ABSTRACT

Keywords: Chitosan Electrostatic spraying Molecular weight Film property

Chitosan (CH) films were prepared with different number-average molecular weight (MW, ca. 6.55, 12.93 and 47.70 kDa) and concentration (0.5, 0.75, and 1.0%) using electrostatic spraying (ES) technique. The effects of MW on film-forming solution properties, ES-atomization performances, and film characteristics were investigated. With increase of MW, conductivity, viscosity, surface tension, and contact angle of CH film-forming solution was raised due to the increases of proportions of amine-groups and degrees of CH chain entanglements. Spray cone angle was generally higher at 12.93 kDa, while the average median diameters of film-forming solution droplets were in the range of 6.3–12.0 μm, indicating well atomization effect. Water barrier property and tensile strength of CH films were improved along with increased MW, which was in accordance with the more completed, rougher and more crystalline microstructures implying by SEM, AFM and XRD. However, film antibacterial capacities against E.coli and L. innocua were descended with increased MW. Considering the overall properties, CH film prepared by 47.70 kDa at 0.5% possessed the best performances.

1. Introduction

performances of the resulted films. Kaya, Asanozusaglam, and Erdogan, (2016) found that the antimicrobial activity of low MW CH (3.22 kDa) was more effective against L. monocytogenes and yeast C. albicans than medium MW CH (ca. 20–80 kDa) at the same DD. Park, Marsh, and Rhim, (2010) evaluated the mechanical, water and oxygen barrier properties of CH films with three different MWs, and reported that the tensile strength of the films increased significantly with the increase of MW while no significant difference was observed for the other properties. However, Bof, Bordagaray, Locaso, and García, (2015) presented that higher MW CH resulted in lower water vapor permeability and color differences of starch-CH films. Hence, the macromolecular characteristics of CH should be considered when preparing films. In order to satisfy the development of new biodegradable films, specific film-forming techniques must be considered to increase efficiency and reduce cost (Peretto, Du, Avena-Bustillos, Berrios, & Sambo, 2017). Electrostatic spraying (ES) is a technology to form coatings on food to enhance the odor, flavor, color, appearance and safety of the product (Barringer & Sumonsiri, 2015). In the ES system, materials are electrically charged and dispersed into tiny droplets/particulates, and the sizes and motions of droplets/particulates are controlled by electric

Food products are easily to be contaminated and perishable by nature, which require protection from spoilage during storage and transportation (Valdés, Ramos, Beltrán, Jiménez, & Garrigós, 2017). Plant-source agents such as essential oils and organic acids, microbial-sources additives including nisin and natamycin, or animal-source agents like lysozyme have been widely applied to inhibit microorganism growth in food product (Valdés et al., 2017). Besides, edible films, made from polysaccharides, proteins, lipids, or the combinations, are widely used as carriers of antimicrobial additives to improve their stability and control the release (Pandey & Ramontja, 2016; Tavassolikafrani, Shekarchizadeh, & Masoudpourbehabadi, 2016). Among the biopolymer materials, chitosan (CH) shows great potential due to its favorable antimicrobial capacity, excellent mechanical characteristics, selective semipermeability to atmosphere gas and nice stabilization capability with its polycationic nature (Pandey & Tiwari, 2015; Pandey, 2017; Sreekumar, Lemke, Moerschbacher, TorresGiner, & Lagaron, 2017; Suyatma, Tighzert, Copinet, & Coma, 2005). It has been reported that the intrinsic properties of CH, such as molecular weight (MW) and degree of deacetylation (DD), could impact the

Corresponding author at: Department of Food Science and Engineering, 800 Dongchuan Road, Shanghai Jiao Tong University, Shanghai 200240, China. Corresponding author at: Department of Food Science and Technology, 100 Wiegand Hall, Oregon State University, Corvallis, OR 97331, USA. E-mail addresses: [email protected] (Y. Zhong), [email protected] (Y. Zhao). 1 The author contributed equally to this work. ⁎

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https://doi.org/10.1016/j.carbpol.2019.02.048 Received 16 May 2018; Received in revised form 12 February 2019; Accepted 14 February 2019 Available online 16 February 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.

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field (Barringer & Sumonsiri, 2015). The main advantages of ES over conventional dipping or spraying method are reducing wastage and pollution, producing homogenous distribution, and enhancing deposition efficiency on object (Gorty & Barringer, 2011; Zhong, Cavender, & Zhao, 2014). CH micro-particles encapsulating bioactive molecules were made by ES process recently and exhibited potential in food packaging (Gómez-Mascaraque, Sanchez, & López-Rubio, 2016; Sreekumar et al., 2017). ES has also been used to prepare stand-alone films. For examples, Stoleru, Munteanu, Dumitriu, Coroaba, and Drobotă, (2016) deposited CH/vitamin E formulation on polyethylene using ES technique to prepare a dual-bioactive layer film. They reported that the film presented antibacterial, antioxidant and pH responsive activity and exhibited good stability. Pareta and Edirisinghe (2006) prepared maize starch films using ES method and pointed out that the times involved in ES process were much shorter than in solvent casting process, and ES ensured even spreading of droplets and uniform thickness of films. However, little is known about the relationship of droplet deposition behavior and film formation properties during ES. According to Barringer and Sumonsiri (2015), powder properties including particle size, flowability, resistivity and density could impact the efficiency of electrostatic powder spraying, while liquid resistivity, viscosity and surface tension were the most essential factors in electrostatic liquid spraying. It has been reported that increasing resistivity and viscosity of liquid chocolate caused the increase of droplet size and the reduction of coating coverage in ES system (Gorty & Barringer, 2011). In order to understand the impact of material properties on droplet deposition behavior during ES and the subsequent film-forming properties, CH films with three MWs and concentrations were prepared by ES in this study. The effects of MW on the properties of film-forming solution, ES efficiencies and physicochemical performances of resulted films were evaluated. The results could provide theoretical guidance to food preservation by CH coating using ES system.

to form films. Briefly, film-forming solution (300–400 mL, with regard to MW) was sprayed onto a horizontal glass plate (250 × 250 mm2) taking about 7–10 min to obtain film with a thickness of ca. 0.04–0.05 mm. During spraying, the spray gun was vertically fixed ca. 35 cm above the plate and the spraying speed, feeding pressure, voltage and load current were controlled at 3.8 L/h, 1.8 kg/cm3, 7.5 kv and 60 mA, respectively. After spraying, the plate was placed at 25 °C and 50% relative humidity chamber (TL-05-040-FH, TPS Tianyu Experimental Equipment Co., Ltd., Chengdu, China) for 48 h to obtain dried film. The dried CH film was carefully pulled off the plate by hand.

2. Materials and methods

2.6. ES performance of the film-forming solutions

2.1. Materials

For evaluating ES performance to form films, spray cone angle and particle size of the film-forming solution droplets were measured. Briefly, spray cone angle during ES was observed and shot using an industrial camera. The images were then processed and the cone angle was quantified by MATLAB program (MathWorks, Natick, USA). For measuring droplet size, the film-forming solution was sprayed onto 26 × 76 mm2 water sensitive paper (Norvatis, Basel, Switzerland) for 0.4–0.5 s. The paper was carefully removed immediately after the spraying process and placed on flat surface for 10 s allowing the color converting from yellow to blue. The water sensitive paper was then observed and analyzed by a Nikon Eclipse E200 microscope (10 × 10 times, Tokyo, Japan), and the particle size ranges were calculated using MATLAB (MathWorks, Natick, USA).

2.5. Characterization of film-forming solutions Self-association of CH in aqueous solution, conductivity, surface tension (ST) and viscosity of film-forming solutions were quantified using the methods described below. Self-association of CH was measured by pyrene fluorescence method (Amiji, 1995) using an F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). Purified pyrene was dissolved in methanol and added to CH solution to a final concentration of 2.0 μM. The emission spectra of pyrene were analyzed in the range of 360–500 nm at an integration time of 1.0 s. The excitation and emission slit openings were 10 and 2.5 nm, respectively. Electrolytic conductivity was carried out with a DDS-307 A conductimeter (INESA Analytical Instrument Co. Ltd, Shanghai, China) at room temperature. Five hundred milliliters of CH film-forming solution was used for each test. ST was determined at 20 °C by an automatic surface tensiometer (K100, KRUSS, Hamburg, Germany) equipped with a Wilhelmy plate. The viscosity of CH film-forming solution was measured by an R/SCC rheometer (Brookfield, Massachusetts, USA) with rotor CC 25. The viscosity values were obtained at 300 1/s of the shear rate.

CH, (C6H11NO4)n, BR, with deacetylation degree of 80% and three average MWs were obtained from Shandong AK Biotech Ltd. (Shandong, China). Tween 80 and methanol, AR, was purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Glycerol and acetic acid, AR, were procured from Shanghai Lingfeng Chemical Reagent Co. Ltd (Shanghai, China). Pyrene, HPLC grade, was purchased from Sigma Chemical Company (St. Louis, USA) and was purified by recrystallization in methanol. 2.2. MALDI-TOF/MS The number-average MW of CH were determined by MALDI-TOF/ MS (MALDI-7090, Shimadzu, Kyoto, Japan) in linear mode at an acceleration voltage of 20 kV using trihydroxyacetophenone (THAP) as the matrix.

2.7. Microstructures of CH films For analyzing the microstructures of CH films, films made of 1% (w/ w) CH solution were selected. Scanning electron microscopy (SEM), atomic force microscope (AFM), and X-ray diffraction (XRD) were observed using the methods described below. A field-emission scanning electron microscope (Sirion 200, FEI, Hillsboro, USA) was used to observe the microstructure of the crosssection of the films, with gold coating for 30 s after the samples were quenched by liquid nitrogen. The surface morphology and roughness of CH films were observed by an atmospheric atomic force microscopy (Multimode Nanoscope IIIA, Bruker AXS, Karlsruhe, Germany) in tapping mode. The crystallinity of CH films (20 × 20 mm2) was determined by a multi-functional X-Ray diffractometer (D8 ADVANCE Da Vinci, Bruker AXS, Karlsruhe, Germany) using Cu-Kα ray in continuous scanning mode. The test conditions were set as follows: 40 kV of tube pressure,

2.3. Preparation of film-forming solution CH was dissolved in 0.5% (w/w) of acetic acid to prepare 0.5, 0.75 and 1.0% (w/w) CH solutions with addition of 30% (w/w CH) glycerol as plasticizer and 5% (w/w CH) Tween 80 as surfactant (Khalid, Javier, Veronique, & Juani, 2008; Zhong & Li, 2011). After stirring for 30 min, pH of the solution was adjusted to 4.5 using acetic acid. Thereafter, the solution was vacuumized at −0.1 MPa (SHZ-D (III), Gongyi Yuhua, Henan, China) for 1 h to remove air bubbles. 2.4. ES of CH film CH solution was sprayed using an ES unit (SC-ET, Electrostatic Spraying Systems Inc, Watkinsville, USA) with nozzle diameter of 3 mm 198

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30 mA of pipe flow, 5°/min of scanning speed and 5–40° of scanning area.

suspension aliquots were taken out and the optical density (OD) at 600 nm was measured by a microplate reader (Type 1510, Thermo Fisher Scientific, Waltham, USA) at 0.5, 1, 2, 3, 5, 8 and 18 h. And the bacterial growth was reflected by OD increase.

2.8. Water activity and water vapor permeability (WVP) of CH films

2.11. Transmission electron microscopy (TEM) of bacterial cells

Water activity (Aw) of the films was determined by a water activity meter (Fast-lab, GBX Scientific Ltd, France). Cup method (test cups made of Plexiglas (inside diameter of 57 mm and inner depth of 15 mm)) was used to measure the WVP according to the method by Zhuang, Jiang, Zhong, Zhao, and Deng, (2018). The cups were filled with 11 mL of deionized water. The films (80 × 80 mm2) were sealed onto the cups and placed in a humidity chamber at 25 °C and 50% relative humidity (TL-05-040-FH, TPS Tianyu Experimental Equipment Co., Ltd., Chengdu, China). The weights of cups were measured every hour for totally 5 h, and WVP was calculated as follows:

The morphological structures of E. coli and L. innocua were examined by TEM (Wang, Chang, Yang, & Cui, 2015). Bacterial suspensions before and after antibacterial test were centrifugated at 8000 rpm for 10 min. The cell pellets were fixed with 2.5% glutaraldehyde in 0.1 mol/L PBS for 8 h and post-fixed with 1% OsO4 in 0.1 mol/L PBS for 2 h. The samples were then dehydrated in a graded series of ethanol and acetone. Thereafter, the samples were immersed in equal parts of acetone and epoxy resin for 1 h, incubated overnight in mixed acetone and epoxy resin (1:2), and embedded in epoxy resin molds at 60 °C for 2 days. After going through ultrathin section and staining, the images of bacterial cells were observed by a transmission electron microscopy (TALOS F200X, FEI, USA).

WVP = m·L / (A·t ·ΔP) where m is the weight of water permeated through the film (g); L is the thickness of the film (mm); A is the permeation area (m2); t is the time of permeation (d), and ΔP is the water vapor pressure difference across the film (kPa).

2.12. Statistical analysis Three replications were carried out for each sample other than mentioned. Data were processed by SPSS Statistics 19 (version 13.0, Statistical Package for the Social Sciences Inc., Chicago, USA) using one-way ANOVA analysis by LSD to evaluate the significant mean difference (p ≤ 0.05).

2.9. Mechanical properties of CH films The mechanical properties involving tensile strength (TS) and elongation at break (EB) were measured by a TA.XTPLUS texture analyzer (StableMicroSystem, Cardiff, UK) according to the method by Zhuang et al. (2018). Film strips (15 × 100 mm2) were fixed between the tensile grips with initial distance of 50 mm. The stretch rate was set as 0.8 mm/s. Ten repetitions were made for each sample. TS and EB could be calculated as follows: TS = Fm / (L·W)

3. Results and discussion The number-average MWs of three CH samples were estimated by MALDI-TOF/MS and the values were 6.55, 12.93 and 47.70 kDa, respectively.

(1)

where Fm is the maximum load (N); L is the thickness of the film (m), and W is the width of the film (m). EB = (lmax − l0) / l0 × 100%

3.1. Properties of film-forming solutions Pyrene (C16H10) is a fluorescent polyaromatic hydrocarbon preferentially located in the hydrophobic domains. For the fluorescence spectrum of pyrene, intensity of peak I (at 372 nm) was enhanced in polar solvent, while peak III (at 384 nm) was stable. The III/I ratio, therefore, reflected the change of the environmental polarity in solution and was always applied in characterization of the aggregation of solute molecules (Amiji, 1995). In present study, the III/I ratio was in the range of 0.53–1.05 (Table 1), which was similar to the data reported by Amiji (1995). The III/I ratio increased with increasing MW and concentration, indicating that the hydrophobic core of CH aggregate was more compact at higher MW or concentration. Electrical conductivity, as the reciprocal of the resistivity, is an important index of the charge conduction ability for electrolytic

(2)

where lmax is the distance at break (m); l0 is the original distance (m). 2.10. Antibacterial properties of CH films Antibacterial test against E. coli (CICC 10003)and L. innocua (CICC 10297) was conducted using liquid culture method (Zhuang et al., 2018). Briefly, E. coli was inoculated into Luria-Bertani broth (LB) and L. innocua was inoculated into Tryptic soy broth (TSB), respectively, to exponential phase of growth for subsequent test. Film pieces (0.05 g) were added into 50 mL of the bacterial suspension and incubated at 37 °C in a vibrator at 120 rpm for 15 h. Two hundred microliter of

Table 1 Properties of chitosan (CH) film-forming solutions at different molecular weights (MW) and concentrations. Sample

Peak III/I ratio

6.55 kDa-1+ 6.55 kDa-2 6.55 kDa-3 12.93 kDa-1 12.93 kDa-2 12.93 kDa-3 47.70 kDa-1 47.70 kDa-2 47.70 kDa-3

0.53 0.55 0.58 0.54 0.58 0.62 0.87 0.93 1.05

± ± ± ± ± ± ± ± ±

0.01Aa 0.01Ab 0.01Ac 0.01Aa 0.01Bb 0.01Bc 0.01Ba 0.00Cb 0.01Cc

Conductivity (μS/cm)

Surface tension (mN/m)

747.67 ± 3.06Aa 988.67 ± 3.51Ab 1154.33 ± 1.53Ac 798.00 ± 2.00Ba 1028.67 ± 3.51Bb 1216.67 ± 2.08Bc 833.67 ± 4.73Ca 1077.67 ± 1.53Cb 1257.67 ± 0.58Cc

35.42 36.01 36.91 36.50 37.00 37.57 37.08 37.58 38.24

± ± ± ± ± ± ± ± ±

0.03Aa 0.03Ab 0.03Ac 0.03Ba 0.03Bb 0.03Bc 0.03Ca 0.03Cb 0.03Cc

+

Viscosity (pa·s)

Contact angle on glass plate (°)

ND ND ND ND 0.01 0.03 0.01 0.03 0.04

27.30 41.92 51.38 34.45 49.15 52.24 48.18 53.20 54.29

± ± ± ± ±

0.00 0.00 0.00a 0.00b 0.00c

± ± ± ± ± ± ± ± ±

1.64Aa 1.78Ab 1.43Ac 1.87Ba 3.42Bb 1.16Bc 2.91Ca 2.10Cb 1.69Bb

Contact angle on polyethylene plate (°) 61.74 64.78 70.82 70.79 71.21 71.34 75.73 77.72 78.19

± ± ± ± ± ± ± ± ±

2.30Aa 2.94Ab 2.30Ac 1.94Ba 1.42Ba 1.49Aa 2.18Ca 2.28Cab 2.23Bb

1 = CH with concentration of 0.50%, 2 = CH with concentration of 0.75%, 3 = CH with concentration of 1.00%. Different capital letters at the same concentration indicated differences at p ≤ 0.05 level were significant, different lowercase letters at the same MW indicated differences at p ≤ 0.05 level were significant.

++

199

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Fig. 1. ES performance of CH film-forming solution. (a) Spray angle, (b) Box chart of droplet particle size.

solution. As seen in Table 1, the conductivity (κ) of CH solutions was varied in 748–1258 μS/cm. It was reported that the conductivity of CH solution aggrandized from 400 to 3500 μS/cm with concentration increasing from 0 to 1.5% as dissolved in 0.1 M acetic acid (Li, Song, Yang, & Fan, 2006). The conductivity was linearly increased with the concentration, with R2 > 0.98, while the growth rate at high MW decelerated. For acetic acid, there were hydrions, dissociated carboxylic acidic ions and undissociated carboxylic acid molecules in aqueous solution. When CH dissolved, amino groups of CH first bound dissociated hydrions which resulted in the slight decrease of conductivity (Li et al., 2006). With the increase of CH concentration, the dissociated hydrions were bound completely, and more CH molecules began to interact with undissociated carboxylic acid molecules by which charge were transported, and then solution conductivity increased (Li et al., 2006). Given a constant number of the chains, higher MW meant more amine groups, and thus the alkalinity of CH molecule was slightly enhanced. For example, the initial pH values of CH solutions at 1% concentration prepared by 0.5% acetic acid were 4.75, 4.85, and 4.90, respectively, when MW increased from 6.55 to 47.70 kDa. Subsequently, more acetic acid was needed at high MW to obtain the final pH of 4.5, and the solution conductivity enhanced. Surface tension (ST) is an elastic tendency of the fluid surface to antagonize external forces (Zhong et al., 2014). ST values of the filmforming solutions composed of surfactant Tween 80 were in the range of 35–40 mN/m (Table 1). It was reported that the ST of 1.5% CH solution without surfactant was 61.5 mN/m (Choi, Park, Ahn, Lee, & Lee, 2002). And the addition of Tween 80 remarkably decreased the ST in present study. Moreover, the ST showed an upward trend with the increase of MW and concentration, similar conclusion drawn by Gao and Wan (2006). According to the surface tension component theory, ST of CH solution was mainly determined by hydrogen bonding, hydrophobic interaction and electrostatic interaction (Gao & Wan, 2006). For a specific MW, more CH approached each other and more inter-molecular hydrogen bonding and hydrophobic interaction were formed as solution concentration increased. In addition, electrostatic interaction enhanced with concentration as well (as seen by conductivity data, Table 1). Consequently, cohesive energy of CH molecules enhanced which led to increased ST (Gao & Wan, 2006). For a specific concentration, higher MW was easier to aggregate (as seen by pyrene fluorescence data, Table 1) and had more inter-molecular interactions (Gao & Wan, 2006), which resulted in the increase of cohesive energy and thus larger ST value. Viscosity is affected by concentration, MW and the structure of the polymers (Ziani, Henrist, Jérôme, Aqil, & Maté, 2011). It was observed from Table 1 that the viscosity of each CH solution at 6.55 kDa or 0.5% CH solution at 12.93 kDa was too thin, and the value could not be

measured. As for viscosity of 47.70 kDa CH solutions, it was apparent that rising concentration led to higher viscosity. Contact angles of solution reflect its wettability on different substrates. It could be seen from Table 1 that all the contact angles were lower than 90°, indicating that CH solution with Tween 80 could spread on both the hydrophilic and hydrophobic surfaces. Specially, the contact angle values on glass were below 55°, exhibiting the better hydrophilic character. Moreover, the contact angle on each substrate raised when MW and concentration increased. As ST values of filmforming solution became larger at higher MW and concentration (Table 1), the solution was harder to spread on substrate which was indicated by higher contact angle. 3.2. Spray cone angle and droplets particle size of film-forming solutions during ES process In order to obtain the atomization effect of film-forming solution during ES process, the spray cone angle was measured and is listed in Fig. 1. + 6.55 = CH with MW of 6.55 kDa, 12.93 = CH with MW of 12.93 kDa, 47.70 = CH with MW of 47.70 kDa; 1 = CH with concentration of 0.50%, 2 = CH with concentration of 0.75%, 3 = CH with concentration of 1.00%. It could be seen from Fig. 1 that the spray angles of CH solution at 12.93 kDa were generally higher, while the values were the least at 47.70 kDa. Especially, the spray angle of 1.0% solution at 47.70 kDa was nearly half of the value at 12.93 kDa. As for the impact of concentration, the spray angles significantly (p ≤ 0.05) increased with concentration for solution at 6.55 kDa, while the spray angle values of 0.75% concentration were the largest for solutions at other MWs. Barringer and Sumonsiri (2015) stated that viscosity, surface tension and electrical resistivity were major factors of solution to affect the ES performance. It had been found that the increase in the fluid viscosity led to decreased area coverage which might be caused by poorer charge diffusivity (Maski & Durairaj, 2010). When the solution resistivity enhanced, the repulsion of droplets became weakened after leaving the electric field, and thus the coating area decreased (Gorty & Barringer, 2011). The solution could not be atomized when the conductivity was outside of the range of 10−8-10-5 S/m (Abu-Ali & Barringer, 2005). In present study, CH solution at low MW or concentration was watery and the impact of conductivity was prevailed, while the influence of viscosity enhanced when MW or concentration increased, which led to different changes of spray angles. Droplet particle size was determined and displayed in Fig. 1. The particle sizes were in the range of 4–26 μm and the average median diameter was 6.3–12.0 μm. It was reported that the droplet diameters of 200

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Fig. 2. SEM images of CH films by ES system with different MW at concentration of 1%.

CH solution with viscosity of 0.077 pa·s and surface tension of 71 mN/m were in the range of 7–33 μm and the mean diameter began to fall as ST was reduced (Pancholi, Ahras, Stride, & Edirisinghe, 2009), which was comparable with the droplet diameters in our study. Researches pointed out that no significant correlation could be found between droplet size and electrical resistivity as well as viscosity in non-electrohydrodynamic (EHD) system (Marthina & Barringer, 2012), while the chocolate with higher resistivity had larger droplet size in EHD system (Gorty & Barringer, 2011). Sumonsiri and Barringer (2010) indicated that there was a decrease in adhesion when powder particle size increased, resulting in lower side coverage of powder materials.

Based on the XRD patterns in Fig. 4, 6.55 kDa CH film possessed two major diffraction peaks at 2θ value of ca. 10° and 20°, which was similar with CH powder (Epure, Griffon, Pollet, & Avérous, 2011). The peak at 10° was attributed to the hydrated crystals while the peak at 20° was related to the regular crystal lattice of CH (Epure et al., 2011). Moreover, there appeared new peaks at around 15° and 17.5° in 12.93 and 47.70 kDa films, which were assigned to a metastable hydrated-Form-II crystalloid (Shariatinia & Fazli, 2015). Macromolecular polymers could crystallize in chain-folded or chain-twisted fashion (Zen et al., 2006). As CH displayed higher tendency of molecular assembling and self-aggregation at increased MW (as seen by III/I ratio of pyrene peak), the interactions of chain segments were enhanced during drying and the crystallinity of the film was then accelerated (Pang, Chen, Park, Dong, & Kennedy, 2007), which in accordance with AFM data in Fig. 3.

3.3. Microstructure of CH films The morphology of cross section of CH film with different MW is shown by SEM (Fig. 2). The cross-sectional structure was relatively flat and uniform. However, there appeared some tiny holes in 6.55 and 12.93 kDa films and the diameters of the holes were in the range of 25–90 nm. Moreover, there were almost no holes in 47.70 kDa film and the film was more integrated. Charged droplet particles were mainly subject to gravitation, electric field force and air force in ES system (Barringer & Sumonsiri, 2015). After leaving the spray gun, the particles were deposited on target surface under actions of the above forces. There existed electrostatic forces between the charged droplets in air and the already deposited charged layer, which was important for droplet deposition on target surface. The droplets could randomly deposit, or repulsively deposit on target (gap filling deposition) which meant incoming droplets filling the empty gaps (Mki, Mai, Schroën, & Boom, 2012). It was found that droplets followed random deposition trajectory on conductive target, while deposition pattern of droplets was closer to gap filling on surface with intermediate conductive property between aluminium foil and Parafilm (Mki et al., 2012). As the conductivity of CH solution became higher with MW increased, more effective coverage and complete film formation without pinholes was obtained (Abu-Ali & Barringer, 2005; Gorty & Barringer, 2011; Khan, Schutyser, Schroën, & Boom, 2012). Furthermore, more entanglements of CH chains at high MW (Gómez-Mascaraque et al., 2016) might also promote the integrity of resulted film. The AFM ichnography and topography images in Fig. 3 revealed the surface morphologies of CH films. The films were unevenly distributed and appeared some aggregations. Surface topography indices including Ra and Rmax could describe surface roughness (Rodriguez, Autio, & Mclandsborough, 2008). In this study, the film at 6.55 kDa had the lowest Ra (7.558 nm) and Rmax (84.530 nm), while greater surface roughness values were obtained in 12.93 and 47.70 kDa films, with the Ra of 18.432 and 14.787 nm, and Rmax of 118.38 and 272.37 nm, respectively. With increasing of MW, the frequency of CH chain entanglement and the intermolecular cohesion in solution enhanced (Gómez-Mascaraque et al., 2016). Moreover, the spray angle decreased with MW (Fig. 1) which meant more CH solution deposited on the same area, promoting the contact of CH molecular and the chain entanglement. Subsequently, film became rougher after drying when MW increased.

3.4. Water activity (Aw) and water vapor permeability (WVP) of CH films Water activity of CH film is displayed in Table 2. The Aw values were generally less than 0.60 except for the 0.50% of 6.55 kDa film. Although all films were conditioned at the same conditions, Aw decreased with the increasing MW and concentration, implying less water absorbing capacity of CH films. It was seen from Table 2 that the water barrier abilities of the films were much better at high MW and the WVP values of 6.55, 12.93 and 47.70 kDa film were 73–80, 49–58 and 26–47 g·mm/(m2·d·kPa), respectively, which were similar with values of CH films prepared by casting method (Bof et al., 2015). It was reported that water vapor transfer depended on both water diffusivity and solubility in a polymeric matrix (Zhong & Li, 2011). As the matrix absorbed less water (as seen by Aw) and was more complete (as seen by SEM) at high MW, the WVP was lower. Furthermore, there was a negative linear correlation between concentration and WVP, indicating higher water diffusivity at high concentration. Escárcegagalaz, Sánchezmachado, Lópezcervantes, Sanchessilva, and Maderasantana, (2018) also reported that WVP of CH film raised from 0.14 g/kPa·h·m2 to 0.33 g/kPa·h·m2 when solution concentration varied from to 1% to 3% due to the hydrophily of CH. 3.5. Mechanical property of CH films The mechanical performance including TS and EB are summarized in Table 2. The TS values were in the range of 8.7–22.6 MPa and EB ranged from 17.5% to 49.7% in present study, which were comparable with values of CH films prepared by conventional method (Bof et al., 2015). The TS values of the films increased as MW increased, and the rate slowed down at high concentration, which was in agreement with the study by Velickova, Winkelhausen, Kuzmanova, Moldão-Martins, and Alves, (2015). According to the results of microstructures, the films became more complete (SEM), self-aggregate (AFM) and crystalline (XRD) at larger MW, and subsequently the mechanical strength enhanced (Suyatma et al., 2005). In addition, the TS value linearly decreased as concentration raised. In order to obtain film with similar thickness (0.04-0.05 mm), the dosage of film-forming solution used for 201

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Fig. 3. AFM images of 1% CH based films by ES system with different MW (scale: 5 μm).

compared with the control group. It could be seen that the inhibition rates of CH films against E. coli were approximately 60% at 5 h with non-significant differences among treatments. The OD growth of L. innocua suspensions with addition of CH film was obviously slower than that of E. coli during experiment, and the value even decreased after 3 h for suspension with 6.55 kDa film. Chen and Zhao (2012) also reported that CH film had a more persistent inhibitory effect against L. innocua than against E. coli. Moreover, the antibacterial capacity of the film was generally reduced with the increase of MW and the decrease of concentration. As for the impact of ES, there seemed that ES did not influence the antibacterial ability obviously. The intact bacterial cells of E. coli and L. innocua, and cell-structures after 24 h of exposure to CH film with 6.55 and 47.70 kDa at 1% concentration were examined by TEM (Fig. 6). It was showed that the initial cells displayed smooth and compact surfaces without any release of intracellular components or notable ruptures on the surfaces. The morphological structures of E. coli were generally retained, but the cell membranes gradually detached from the cell walls and there appeared a few black spots on cell surfaces. The detachment phenomenon was more obvious when E.coli was treated with CH at 6.55 kDa. As for treated L. innocua sample, the integrity of the cell layer structure was significantly destroyed, leading to leakage of intracellular material. The tightly condensed substances or dense granules were assumed to be caused by condensed DNA and cytoplasmic proteins of inactivated cells (Wang et al., 2015). And CH with lower MW brought about more significant damages. In the case of gram (−) bacteria, CH could chelate

Fig. 4. XRD images of 1% CH based films by ES system with different MW.

ES was proportionally reduced at high concentration, which resulted in the weak mechanical resistance. For instance, TS expanded 1.4 times from 6.55 to 47.70 kDa at 1% concentration, while TS reduced 50% from 0.5 to 1% concentration at MW of 47.70 kDa. As for EB, the values generally decreased with MW increasing, while there were no obvious rules of the impact of concentration. 3.6. Antibacterial property of CH films The OD curves of bacteria suspensions at 600 nm are shown in Fig. 5. The addition of CH films significantly inhibited the OD growth

Table 2 Macro properties of CH films at different MW and concentrations prepared by electrostatic spraying (ES) system. Sample

Aw++

6.55 kDa-1+ 6.55 kDa-2 6.55 kDa-3 12.93 kDa-1 12.93 kDa-2 12.93 kDa-3 47.70 kDa-1 47.70 kDa-2 47.70 kDa-3

0.611 0.596 0.587 0.584 0.566 0.544 0.549 0.536 0.525

± ± ± ± ± ± ± ± ±

WVP (g·mm/m2·d·kPa) 0.001Cc 0.002Cb 0.002Ca 0.002Bc 0.003Bb 0.003Ba 0.002Ac 0.003Ab 0.003Aa

73.36 78.00 80.02 49.04 53.54 57.51 26.80 39.17 46.77

± ± ± ± ± ± ± ± ±

2.74Ca 6.21Cb 6.81Cb 1.36Ba 9.64Bab 3.79Bb 2.13Aa 7.26Ab 5.57Ac

+

TS (MPa)

EB (%)

15.77 ± 2.52Ab 12.15 ± 2.07Ab 8.67 ± 1.72Aa 17.68 ± 5.27Bb 14.41 ± 2.29Aa 12.05 ± 2.24Ba 22.64 ± 2.71Cc 17.52 ± 1.88Bb 11.51 ± 2.25Ba

32.83 49.68 32.53 32.80 22.28 35.52 17.52 35.37 25.74

± ± ± ± ± ± ± ± ±

5.57Ba 5.37Cb 4.78Ba 2.00Bb 4.24Aa 6.32Bc 2.62Aa 6.54Bc 3.69Ab

1 = CH with concentration of 0.50%, 2 = CH with concentration of 0.75%, 3 = CH with concentration of 1.00%. Aw: Water activity; WVP: Water vapor permeability; TS: Tensile strength; EB: Elongation at break. +++ Different uppercase letters at the same concentration indicated differences at p ≤ 0.05 level were significant, different lowercase letters at the same MW indicated differences at p ≤ 0.05 level were significant. ++

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Fig. 5. Antibacterial properties of CH films by ES system with different MW against E.coli (a) and L.innocua (b). + 1 = CH with concentration of 0.50%, 2 = CH with concentration of 0.75%, 3 = CH with concentration of 1.00%.

with different cations of the outer membrane or electrostatically interacted with the anionic parts of the lipopolysaccharide, and disturbed the uptake of important nutrients (Verlee, Mincke, & Stevens, 2017). For gram (+) bacteria, CH bound non-covalently with teichoic acids, disrupted their functions, and led to cell death (Verlee et al., 2017).

When the film pieces were added into bacterial suspension, higher concentration of CH film at 6.55 kDa was dissolved to work on the bacterial, and lower MW prompted the permeability of CH through cell membrane as well (Verlee et al., 2017), which both aggravated the cell damages.

Fig. 6. Observation of cell morphology change by TEM. Control pathogen: E. coli (a) and L. innocua (b); treatment with CH film with 6.55 kDa at 1% concentration: E. coli (c) and L. innocua (d); treatment with CH film with 47.70 kDa at 1% concentration: E. coli (e) and L. innocua (f). 203

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In order to comprehensively evaluate the performances of CH film by ES, adhesiveness of film-forming solution (contact angle), ES effects (spray cone angle and droplet particle size) and micro-properties (WVP, TS and antibacterial ability) were selected. The results indicated that CH at 0.5% concentration had better performances, especially at 47.70 kDa. We prepared CH film at 47.70 kDa and 0.5% concentration using traditional casting method as well, and the WVP, TS and EB values of casted film were 24.18 g·mm/m2·d·kPa, 19.90 MPa, and 39.93%, respectively, similar with the values of ES-prepared film. However, ES technology displayed more advantages of industrialized continuous production.

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4. Conclusion Macro properties of CH film prepared by ES system were similar with the properties of CH film prepared by traditional casting method, but ES technology is more suitable for continuous production. As MW increased, the proportion of amine group in a molecule grew, the probability of molecular interaction promoted, and self-entanglement of CH chain enhanced, which led to higher III/ I ratio of pyrene fluorescence, viscosity, conductivity and surface tension of film-forming solution. The aforementioned solution properties impacted droplet motions in electric field, and thus the spray cone angle values were generally higher at 12.93 kDa. With the influence of charge transfer and molecular chain entanglement, a more complete, self-aggregate and crystalline film formed when MW increased. Consequently, the CH film possessed much lower WVP and higher TS at higher MW. The antibacterial capacities of the films against E. coli and L. innocua were improved with MW declining, and the influence of ES was not obvious. Considering the overall spraying effects and macro-properties of the resulted films, CH with 47.70 kDa at 0.5% concentration displayed the best performance. Molecular modeling is a convenient technique to predict the behaviors of polymers in solution and interactions of molecules. Further research was needed to simulate and predict the CH molecular behavior in solution using molecular simulation and mesoscopic simulation methods. Conflict of interest All authors have no competing interests to disclose. Acknowledgements This work was supported by the National Natural Science Foundation of China [Nos. 31501533], Grant from the Wilmar (Shanghai) Biotechnology Research & Development Center Co., Ltd. [Nos. 0A16040007123], and SJTU startup fund for young talent [Nos. 15X100040023]. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2019.02.048. References Abu-Ali, J., & Barringer, S. A. (2005). Method for electrostatic atomization of emulsions in an EHD system. Journal of Electrostatics, 63(5), 361–369. Amiji, M. M. (1995). Pyrene fluorescence study of chitosan self-association in aqueous solution. Carbohydrate Polymers, 26(3), 211–213. Barringer, S. A., & Sumonsiri, N. (2015). Electrostatic coating technologies for food processing. Annual Review of Food Science and Technology, 6(1), 157–169. Bof, M. J., Bordagaray, V. C., Locaso, D. E., & García, M. A. (2015). Chitosan molecular weight effect on starch-composite film properties. Food Hydrocolloids, 51, 281–294. Chen, J. L., & Zhao, Y. (2012). Effect of molecular weight, acid, and plasticizer on the physicochemical and antibacterial properties of β-chitosan based films. Journal of Food Science, 77(5), 127–136. Choi, W. Y., Park, H. J., Ahn, D. J., Lee, J., & Lee, C. Y. (2002). Wettability of chitosan

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