Chitosan-sodium phytate films with a strong water barrier and

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Chitosan-sodium phytate films with a strong water barrier and antimicrobial properties via one-step consecutive stripping and layer-by-layer casting technologies Jie Yang, Liu Xiong, Man Li, and Qingjie Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01890 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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Journal of Agricultural and Food Chemistry

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Chitosan-sodium phytate films with a strong water barrier and

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antimicrobial properties via one-step consecutive stripping and

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layer-by-layer casting technologies

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Jie Yang1, Liu Xiong1, Man Li, Qingjie Sun*

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College of Food Science and Engineering, Qingdao Agricultural University (Qingdao,

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Shandong Province, 266109, China)

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1

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*Correspondence author (Tel: 86-532-88030448, e-mail: [email protected])

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College of Food Science and Engineering, Qingdao Agricultural University, 266109, 700

Equally-contributing author

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Changcheng Road, Chengyang District, Qingdao, China.

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ABSTRACT：： The pursuit of sustainable functional materials requires the development of

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materials based on renewable resources and efficient fabrication methods. Hereby, we first

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fabricated chitosan-sodium phytate films via one-step stripping and layer-by-layer casting

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technologies, respectively. The proposed film fabrication methods are general, facile,

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environmentally benign, cost-effective, and easy to scale up. The resultant one-step stripping film

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was thin (9±1 µm), soft, transparent, and strong, while the thickness of the layer-by-layer casting

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film was 70±3 µm. FTIR analysis of the films indicated the formation of interactions between the

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phosphoric groups in sodium phytate and the amino groups in chitosan. More importantly, the

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water vapor permeability values of one-step stripping and casting films were 4-5 orders of

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magnitude lower than chitosan films reported before. Layer-by-layer casting films in particular

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exhibited high tensile strength (49.21±1.12 MPa), which were more than three times stronger than

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that of other polyelectrolyte multilayer films. Both types of films remained stable in an acidic

ACS Paragon Plus Environment


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environment. Meanwhile, the layer-by-layer assembly films presented greater antimicrobial

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activity than the stripping films. The developed chitosan-sodium phytate films can enhance

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several biomedical and environmental applications, such as packaging, drug delivery, diagnostics,

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microfluidics, and biosensing.

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KEYWORDS: water vapor permeability, swelling, tensile strength, stability, coating

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INTRODUCTION

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Due to the drawbacks of petroleum-derived bio-stable plastics, researchers have recently

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turned utilizing different kinds of biopolymers to prepare film.1 Generally, synthetic polymers

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were used to enhance the barrier properties of films; however, the increased use of synthetic

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polymers has raised serious concerns on account of their serious recycling and environmental

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challenges.2 Nowadays, it is of commercial interest to develop natural, sustainable, renewable, and

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functional films. 3,4 In general, biopolymers derived from natural products, like polysaccharides

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and proteins, are considered ideal candidates to replace synthetic polymers for various

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applications because of their low cost, the renewability, biodegradability, and biocompatibility of

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their sources, and their non-toxic, environmentally friendly processes.5 Lately, chitosan, an

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electropositive biopolymer, has attracted widespread interest in the field of food coating and

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wrapping materials due to its non-toxic, biocompatible, biodegradable, antimicrobial, and good

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film-forming properties.6

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However, the use of pure chitosan films has thus far been limited because they tend to be

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rigid, brittle, and have poor water resistance, low mechanical strength, and so on.7 Thus, several

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studies have been conducted, mostly in the last decade, to modify chitosan films both physically

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and chemically. For example, many chemical crosslinkers, such as formaldehyde, glyoxal,


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glutaraldehyde, and genipin, have been investigated to improve film wettability, elongation at

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break, and thermal stability compared to pure chitosan film.8 Nevertheless, these crosslinkers are

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evermore associated with a negative environmental impact. Porta et al.9 reported that the

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chitosan-based edible films crosslinked with transglutaminase could create environmentally

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friendly bioplastics. But it could not meet industrial demand due to its high cost and low efficiency.

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Hence, developing biodegradable films with a low cost, high water vapor barrier, and enhanced

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mechanical properties on a large scale is still a critical issue.

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One major research direction has focused on the water vapor barrier properties and tensile

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strength of natural chitosan-sodium phytate films, which can expand the application scope to a

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significant extent. This study was inspired by a previous study on the preparation of

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chitosan-sodium phytate nanoparticles through strong electrostatic and hydrogen bond interactions

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between the phosphoric groups in sodium phytate and the amino groups in chitosan.10 Sodium

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phytate consisted of six phosphate groups and was chosen as the negatively-charged polyanions,

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as well as being widely used as a safe food additive extracted from natural raw materials, such as

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plant bran and seeds. Importantly, sodium phytate shows excellent antibacterial activity and is

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listed as a “generally recognized as safe” (GRAS) substance by the Food and Drug Administration

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of the United States.11 Until now, only a few studies have reported that phytic acid is successfully

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assembled into layer-by-layer films with chitosan.12 However, to the best of our knowledge,

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sodium phytate has not previously been used to prepare chitosan films.

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Generally, the film’s properties are closely connected with the type of preparation method

65

that is used. In this work, we prepared chitosan-sodium phytate films using two types of facile

66

methods, one-step consecutive stripping and layer-by-layer (LBL) casting technologies.



67

Commonly, one-step stripping film is formed at the solution interface with the key catalyst (such

68

as abundant oxygen molecules)13 or high temperature.14 Interestingly, our chitosan-sodium phytate

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film can be prepared at room temperature without adding anything else. The LBL casting

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technique is also a simple, easy, versatile, flexible, handling, inexpensive, and powerful tool for

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creating stratified and versatile multilayer films with strong mechanical properties, based on

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interactions between multivalent molecules and macromolecules.15 The technique has a number of

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advantages, for example, the film thickness can be controlled with an almost defect-free structure;

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Secondly, it is very versatile and can accommodate various components.16 We systematically

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investigated the formation, physicochemical properties, stability, mechanism of film formation,

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and antimicrobial activity of the chitosan-sodium phytate films. Such chitosan-sodium phytate

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films may be recommended for different applications, including food, medical sciences, and drug

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delivery.

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MATERIALS AND METHODS

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Materials：：Chitosan was purchased from Shanghai Ryon Biological Technique Co., Ltd.

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(Shanghai, China). The viscosity-average molecular weights of chitosan were 1092±50 kDa and

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the degree of chitosan deacetylation was approximately 90%. Sodium phytate was obtained from

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Beijing Solarbio Science & Technique Co., Ltd. (Beijing, China). Urea was provided by

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Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Staphylococcus aureus (S. aureus, ATCC

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25923) and Escherichia coli (E. Coli, ATCC 25923) were acquired from Nanjing Bianzhen

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Biological Technology Co., Ltd. Lysogeny broth (LB) powder was supplied by Thermo Fisher

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Scientific Inc. (Beijing, China). All chemicals were of analytical grade and used without further

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purification.


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Film Fabrication

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Film-stripping technique. Film-stripping technique: Chitosan was dissolved in 2.0 wt%

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glacial acetic acid to prepare 2.0-5.0 wt% solution. The concentration of sodium phytate solution

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was 0.625-4.0 wt%. Chitosan solution was first added into a beaker and then sodium phytate

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solution was added on the surface of the chitosan solution and promptly smoothed out. The

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volume ratio of chitosan to sodium phytate was 5:1. Then, the chitosan-sodium phytate film was

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spontaneously formed rapidly within 1 min at the chitosan/sodium phytate interface without

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stirring. Only the part of chitosan contacting with the sodium phytate solution was converted into

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film. The film was stripped using tweezers and washed with water three times to remove excess

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chitosan and sodium phytate. After the first film was finished, sodium phytate solution left on the

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surface of the chitosan solution was removed. Then, pouring the sodium phytate solution into the

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surface of the chitosan solution spontaneously formed the second film. The second film was

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stripped and washed with water as mentioned above. This stripping process could be repeated

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continuously. Finally, the wet film was allowed to air-dry on a glass pane.

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Layer-by-layer casting technique. The chitosan/sodium phytate multilayer films were

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prepared based on the LBL casting technique described previously.17 A 20 mL of 2.0-5.0 wt%

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chitosan solution was first cast on a glass plate, forming the first layer. Subsequently, another 5

106

mL of sodium phytate solution (0.625-4.0 wt%) was cast on top of the previous chitosan layer. By

107

repeating this process, chitosan/sodium phytate multilayer films consisting of 10 layers were

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prepared. All films were easily peeled off from the supporting petri dish after they were dried in an

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oven (60ºC). The corresponding films with n layers were denoted as (chitosan/sodium phytate)n.

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All the films were stored in a conditioning room (25ºC and 75% RH) for 3 days before further



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characterization.

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Characterization. The light transmittance of the films was monitored using an ultraviolet

113

(UV)-visible Shimadzu 1601 PC spectrophotometer (Tokyo, Japan) by scanning from 260 to 360

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nm.18 The films were cut into strips (1 × 4 cm2) and placed directly into the test cell of the

115

spectrophotometer. The opacity was calculated by the following equation:

A χ

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Opacity =

117

where A was the absorbance of film at 600 nm and x was the film thickness (mm).

118

The film thickness of film was determined as the average of 5 random positions of each

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sample using a thickness meter (MP0, Fischer, Germany). The zeta potential (ζ potential) was

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determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano (Malvern

121

Instruments Ltd., UK). The measurements were performed on samples diluted in MilliQ water at

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25ºC. X-ray diffraction (XRD) measurement was conducted on an X-ray diffractometer (Bruker

123

D8 ADVANCE, Karlsruhe, Germany). The diffractograms were recorded over an angular range

124

(2θ) of 4–40º, with a step size of 0.02º and a step rate of 2 s per step. Fourier transform infrared

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spectroscopy (FTIR) was performed on a Nicolet 6700 FTIR spectrometer (Thermo Scientific,

126

USA) at a resolution of 4 cm-1 in the range of 400-4000 cm-1 by cumulating. The water vapor

127

permeability (WVP) of the films was determined gravimetrically.19 Before testing, the films were

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conditioned at 25ºC for 3 days in a desiccator with a relative humidity (RH) of 75%. The film

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specimen was sealed on a circular dish (diameter of 30 mm) filled with 15 g of oven dried calcium

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chloride (CaCl2) that can maintain 0% RH in the circular dish. The circular dish was kept in the

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test chamber and weighed every 12 hours for 3 days. The amount of water vapor that penetrated

132

through the film was determined by the weight increase of the circular dish. The WVP was

(1)


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calculated as follows:

WVP(g / m ⋅ h ⋅ Pa ) =

m×d A×t×P

(2)

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where m is the weight increment of the cell (g), d is the film thickness (m), A is the area exposed

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(m2), t is the time lag for permeation (h), and P is the water vapor partial pressure difference across

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the film (Pa).

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The gravimetric method was used to determine the water solubility and degree of swelling of

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the films.20 The weight of the film strips (1×4 cm2) was determined (m1). Then, the samples were

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dried at 105ºC for 24 h to obtain the dry mass (m2). The samples were submerged in distilled

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water at 25ºC for 24 h and achieved a constant mass (m3). Then, the samples were dried at 105ºC

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for 24 h, after which the dry weight of the sample was determined (m4). The water solubility,

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degree of swelling, and moisture content were calculated as follows:

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m2 − m4 m4 m3 − m 2 Degree of swelling (%)= m2 m1 − m 2 Moisture content (%)= m1

Water solubility (%)=

(3) (4) (5)

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Thermal analyses (TGA) of films were measured simultaneously by TGA/DSC2 (METTLER

148

TOLEDO, Greifensee, Switzerland). Approximately 3 mg of films were heated from 30 to 600ºC

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at a heating rate of 10ºC/min. The weight loss as a function of temperature was analyzed.

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The mechanical properties of one-step stripping film, which was somewhat soft, were

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executed using a texture analyzer (TAXTplus, Stable Micro Systems, Surrey, U.K.), fitted with an

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A/SPR probe. The test speed was 3 mm/s. The strong LBL casting film was determined on an

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electronic universal testing machine (WDW3100, Changchun, China) at room temperature. The

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samples were cut into rectangular strips (10 mm × 100 mm). The clamp distance was 20 mm, and



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the draw rate was 20 mm/min. Based on the resultant stress-strain curves, the strength at break,

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Young’s modulus, elongation at break, and toughness were obtained. Before the testing, the strips

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were preconditioned at 75% RH for 3 days at room temperature (25ºC).

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Scanning electron microscopy (SEM) (S-4800, Hitachi Instruments Ltd., Tokyo, Japan) was

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used to study the morphology and topography of the surface and cross sections of the films. The

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films were frozen in liquid nitrogen and then fractured immediately. The surfaces and the fracture

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cross sections were sputtered with gold and then photographed. The acceleration voltage was set

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to 1 kV.21

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Glass transition temperature readings of the films at sub-zero temperatures were performed

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by differential scanning calorimetry DSC1 (METTLER TOLEDO, Switzerland) at the scan rate of

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10ºC/min from -70 to 25ºC in an argon gas atmosphere. Films were dried in a vacuum oven at

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40ºC for 24 h to remove water without changing the solid-state structure. The samples were

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incubated at 25ºC for 3 days under various RHs, which were controlled by the saturated water

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vapor of 8 types of salt, according to a previous method. The 8 salts used in this study were

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lithium chloride (RH 11%), magnesium chloride (RH 33%), potassium carbonate (RH 43%),

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sodium bromide (RH 58%), potassium iodide (RH 69%), sodium chloride (RH 75%), potassium

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chloride (RH 84%), and potassium sulfate (RH 97%).

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pH and temperature stability. The stability of chitosan-sodium phytate films at different

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pH levels and temperatures was determined using a gravimetric method to measure the erosion of

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the films. The dried films were weighed (W0) and soaked in different pH buffer solutions (pH 2-9)

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at room temperature or 4, 25, 37, and 60ºC water at pH 7. The films were removed from the

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medium after 1 h of incubation, blotted using filter paper to remove excess water, and then dried


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in a hot air oven at 60ºC to obtain a constant weight (Wd).22 The erosion of the films was

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calculated according to the following equation:

W0 − Wd ×100 W0

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Erosion (%) =

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The stability of film in different solvents. The erosion of the films was measured to

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determine the interaction force of films between chitosan and sodium phytate. The dried films

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were soaked in each dissociating reagent, including sodium chloride (NaCl, 10, 50, 100, 250, and

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500 mM) and urea (1, 2, 3, 4, and 5 M). The films were removed from the medium after 1 h of

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incubation, blotted using filter paper to remove excess water, and then dried in a hot air oven at

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60ºC to obtain a constant weight. The erosion of the films was determined according to the

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above-mentioned equation (6).

(6)

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Antibacterial property. An aliquot of 300 µL of the bacterial suspension (105 CFU/mL)

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with 2.7 mL of LB broth was transferred into sterile tubes. The film samples were soaked in the

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bacterial solution and incubated at 37°C for 4 h. The final inoculum was recultivated on a solid

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agar plate surface for 24 h. We determined the antibacterial rates by the following equation：

CFUcontrol − CFU exp erimental × 100 CFUcontrol

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Antibacterial rate (%) =

192

where the bacteria only served as the control, while the chitosan-sodium phytate film

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(7)

prepared by the film-stripping and LBL-casting technique comprised the experimental groups.23

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Statistical analysis. Each measurement was carried out using at least three fresh,

195

independently prepared samples. The results were reported as the mean values and standard

196

deviations. The experimental data were analyzed by the analysis of variance (ANOVA) with the

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SPSS V.17 statistical software package (SPSS Inc., Chicago, IL). Differences were considered at a

198

significance level of 95% (p < 0.05).



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RESULTS AND DISCUSSION

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Formation of chitosan-sodium phytate film. The pH of the chitosan solution was 3.0 (ζ

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potential = +45 mV). The sodium phytate was dissolved separately in water and the pH of the

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solution was 11.0 (ζ potential = -13 mV). The film fabrication process of the film-stripping and

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LBL-casting methods is shown in Figure 1. To develop an optimal strategy for the preparation of

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smooth, uniform, strong, and biocompatible chitosan-sodium phytate films with tunable properties,

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the parameters that influence the properties of the films were systematically investigated.

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Characterization of chitosan-sodium phytate films

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The thickness, opacity, and water vapor permeability. The thickness, opacity, and water

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vapor permeability of the films were characterized (Table 1 and Table S1). The thickness of both

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stripping and casting films increased with the increasing concentration of sodium phytate. The

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resultant stripping film was thin with thickness of 9±1 µm, which was thinner than protocatechuic

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acid grafted chitosan films (44.1 µm).24

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The opacity is a critical property when films are applied to food packaging to improve

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product appearance.25 The opacity values of stripping films with incremental sodium phytate

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concentrations ranging from 0.625% to 5% (w/w) led to a significant decrease from 4.556±0.220

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to 2.308±0.102 A600/mm. The LBL films presented the minimum opacity values of 0.992±0.011

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A600/mm at 1.25% sodium phytate. The UV-vis spectrometer was used to investigate the

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transmittance of stripping and casting films (Figure 2A, B, Figure S1). The light transmittance of

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the films decreased sharply at around 315 nm with the increasing concentration of sodium phytate.

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The stripping films possessed remarkably low transparency (61%, at around 315 nm) at the

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sodium phytate concentration of 4.0 wt%. The casting films exhibited a lower transmittance of


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21% at around 324 nm at the sodium phytate concentration of 4.0 wt%. Our findings suggested

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that LBL film provided better protection of food quality from ultraviolet light damage.

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Figure 2 (C and D) shows a few pieces of paper with the logo of Qingdao Agricultural

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University placed under the chitosan/sodium phytate films. The logo can be seen clearly through

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the film. The color of the stripping films did not change significantly (Figure 2C and Figure S2

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A-B); however, the color of the casting films turned a deeper yellow (Figure 2D and Figure S2

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C-E) with the increased concentration of sodium phytate. The obtained stripping films were glossy

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and extremely flexible (Figure 2E), while LBL casting films were tough and pressure-resistant

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(Figure 2F), which was due to strong interactions between chitosan and sodium phytate by

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layer-by-layer technology. The interactions not only greatly enhanced the toughness of the films,

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enabling them to tolerate substantial deformation, but also facilitated optically transparent

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materials.

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Since the stripping film surfaces were different, one side was enriched in chitosan and the

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other was in sodium phytate. Considering the film application as coating materials, generally,

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chitosan is first coated, and then sodium phytate is added on the surface of chitosan. Therefore, we

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chose the surface of sodium phytate to be exposed to the high water activity environment during

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the WVP tests. The WVP value increased with the increasing chitosan concentration. The stripping

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film exhibited a minimum WVP of 1.654±0.062 ×10-11 g/m·h·Pa with 1.25% sodium phytate and

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3% chitosan. Then, the WVP of stripping films increased to 5.085±0.162×10-11 g/m·h·Pa when the

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chitosan concentration increased to 5%, and the maximum WVP occurred with 5% chitosan and

241

5% sodium phytate. However, the casting film exhibited minimum and maximum WVP values of

242

2.150±0.099×10-11

g/m·h·Pa

(with

3%

chitosan

and

0.625%


sodium

phytate)

and


243

23.549±0.918×10-11 g/m·h·Pa (with 5% chitosan and 4% sodium phytate), respectively. This

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tendency could be explained by the higher hydrophilicity (NH3+ and -OH groups) of the films with

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higher concentrations of chitosan and sodium phytate. The WVP values achieved for two kinds of

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chitosan-sodium phytate films were four to five orders of magnitude lower than chitosan-based

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films (3.32 × 10-10 g(m s Pa)-1, ≈1.20×10-6 g(m h Pa)-1).26 Bourtoom and Chinnan 27 reported that

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the WVP value of rice starch-chitosan film increased after the incorporation of chitosan. Bonilla et

249

al.28 also suggested that the WVP value of wheat starch-glycerol films increased with the amount

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of chitosan embedded.

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Water solubility and degree of swelling. Water solubility and the degree of swelling are

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important factors in determining the application of bio-based films. As shown in Figure 3A and B,

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the moisture content (about 20%) of both the stripping and casting films was not affected by the

254

concentration of sodium phytate. However, the water solubility of the stripping films slightly

255

decreased from 21.49% to 15.16% with the increasing of sodium phytate concentration. In water,

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the stripping film showed a decreased swelling degree from 91.63 to 66.34%, but it still

257

maintained its original shape. The water solubility and swelling degree values for the stripping

258

films were much lower than that of polyphenol-chitosan film (19.93±0.76% and 353.92±11.21%,

259

respectively), as reported by Sun et al.29 The outstanding water resistance could be ascribed to the

260

compact structure that hindered the water penetration into the films (by decreasing the permeation

261

of water into the films via capillary force). It's worth noting that for casting films, with the

262

increase of the concentration of sodium phytate, the water solubility continually increased, while

263

the degree of swelling first increased and then increased (Figure S3). Moreover, at the same

264

concentration of sodium phytate, the degree of swelling gradually increased with the increase of


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chitosan concentration. This could be due to the swelling of chitosan in the presence of acetic acid.

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Bourtoom and Chinnan 30 reported that higher solubility indicated lower water resistance.

267

Mechanical properties. To evaluate the potential of films as structural material for industry,

268

the mechanical properties of chitosan-sodium phytate films were characterized. The results of

269

tensile strength, elongation at break, and Young's modulus for films prepared by film-stripping

270

technology are shown in Table S2. The stripping film with the highest tensile strength values was

271

32.44±1.49 MPa. The stress-strain curves of the LBL films obtained from the tensile tests are

272

shown in Figure 3C and Figure S4. The tensile strength and elongation at break values of the LBL

273

films are clearly dependent on the concentration of chitosan and sodium phytate, which yields a

274

maximum tensile strength of 49.21±1.12 MPa at 4.0 wt% chitosan and 1.25 wt% sodium phytate

275

(Table S3), which was higher than that of the stripping film. When the concentration of chitosan

276

was 4.0 wt%, the elongation at break and Young’s modulus of films were 25.10±0.71% and

277

3646.68±146.63 MPa, respectively. Thus, strong interaction and good compatibility between

278

chitosan and sodium phytate could have led to uniform stress transfer through the film, resulting in

279

high mechanical strength. The value of tensile strength for LBL films was stronger than that of

280

transcinnamaldehyde-chitosan films (15-20 MPa). 31 Ho et al. 32 noted the beneficial effect of the

281

electrostatic interactions of opposite charges in composite systems on mechanical properties.

282

Thermal properties. Thermogravimetric analysis (TGA) was performed to illustrate the

283

thermal stability of both the stripping and casting films (Figure 3D-G). It can be seen that all films

284

undergo thermal degradation in three stages. The first stage of weight loss of all the films occurred

285

at up to 80ºC and was attributed to the desorption of physically adsorbed water that accounted for

286

20% of the weight of both samples. Later on, the stripping films suffered a drastic weight loss and



287

deacetylation of chitosan chains up at 244ºC. The casting film with 0.625% sodium phytate began

288

the step of maximum weight loss at 267.3ºC, while 1.25, 2, 2.5, and 3% were shown at 257.4,

289

260.3, 265.9, and 238.9 ºC, respectively. Finally, the weight loss continued due to charring of the

290

degraded backbone and there was almost complete degradation up to 600ºC. This result agreed

291

with some reports in the literature. 33 After the final thermal destruction, it was noteworthy that the

292

residual percentages of the casting films at 600ºC were more than 50%, while the residual

293

percentages of the stripping films were less than 50%. This result suggested that the thermal

294

stability of the casting film was higher than that of the stripping films.

295

Film-forming time of stripping films. When 5 ml of 3.0 wt% chitosan solutions were added

296

with 1 ml 1.25 wt% or 2.5 wt% sodium phytate solutions, the stripping films were immediately

297

formed at the chitosan/sodium phytate solution interface within 60 s. The chitosan solution

298

remained in the upper space of the inverted glass vial (Figure 4A, Figure S5A). Commonly,

299

tripolyphosphate (TPP) was used to prepare chitosan film. However, the chitosan-TPP film

300

exhibited a longer film-formation time of ≈1.5 h (Figure S5B). Compared with chitosan-TPP film,

301

the film-formation time of chitosan-sodium phytate film was shortened by 89%. Thus, the

302

formation time of chitosan-sodium phytate film prepared by film-stripping technology was short

303

as well as an ambient temperature. The possible formation mechanisms of film formed in a

304

chitosan/sodium phytate interface-specific manner may be due to the amidogen and hydroxy of

305

chitosan and the rich presence of the phosphate group and hydroxy of sodium phytate. They could

306

form non-covalent bonds, such as ionic bonds and hydrogen bonds, which assembled a

307

three-dimensional network at the interface of chitosan and sodium phytate.

308

The thickness of stripping film gradually increased from 9±1 µm (1 min) to 122±6 µm (30


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309

min) (Figure 4B), and the appearance and transparency of the film became worse over time. The

310

film opacity also increased significantly with the increasing of time (Figure S6 A). The water

311

solubility and moisture content of stripping films showed no change as the preparation time

312

increased, and the lowest swelling degree of the films was found at the preparation time of 5 min

313

(Figure S6 B). In the UV-vis spectrum, the film transmittance gradually decreased with the

314

increasing of formation time, which indicated that the thicker the film, the stronger the ultraviolet

315

absorption (Figure 4C).

316

The thermal degradation of stripping films formed at different time are shown in Figure 4D

317

and E. All the stripping films had a weight loss of about 10%, which occurred because of the

318

release of water molecules from the films. Then, the weight loss in the temperature range of

319

200-240ºC corresponded to the degradation and deacetylation of chitosan. The thermal

320

degradation temperature of the films prepared at different time was similar. The FTIR spectra of

321

the stripping films prepared in various time frames are shown in Figure S7. The intensity of

322

hydroxyl groups peak at 3000-3500 cm-1 was dramatically stronger as formation time increased,

323

indicating that the interaction between chitosan and sodium phytate became stronger.

324

Stripping numbers of films. The chitosan-sodium phytate films prepared by one-step

325

film-stripping technology have a fascinating feature of consecutively stripping at room

326

temperature. The UV-vis light transmission of stripping films with ten stripping numbers are

327

shown in Figure 5A. The transparency of film slightly decreased at around 325 nm from 81% to

328

80% with the increase of stripping numbers. The thickness of the stripping film gradually

329

increased from 50±1 µm to 69±3 µm. The opacity of the films also increased with the increase of

330

stripping numbers (Table S4). Figure 5B shows that stripping numbers did not influence the



331

moisture content (about 20%) of the films. Moreover, the water solubility and swelling degree of

332

the stripping films only slightly changed after the tenth time.

333

The surface morphology images showed that there were no differences in the external

334

appearance, except for the ninth and tenth time, all of which were very smooth and uniform,

335

indicating that the films could be consecutively stripping (Figure 5C). SEM analysis of the

336

fractured cross-section structure of stripping films revealed there was an increasing thickness as

337

the stripping numbers increased (Figure 5D).

338

Forming the temperature of stripping film. Large differences in thickness at different

339

temperatures were observed (Figure S8 A). The thickness of chitosan-sodium phytate film

340

fabricated at 10, 25, or 80ºC was thin, or less than 50 µm. The stripping films prepared at 4ºC had

341

the lowest opacity (Figure S8 B).

342

The stripping films possessed highly remarkable light transmittance at 25ºC. Above or below

343

this temperature, the light transmittance would decrease sharply at around 315 nm (Figure S8 C).

344

The film-forming temperature did not have an impact on the moisture content (about 20%) of the

345

films (Figure S8 D). However, the water solubility of the stripping films increased significantly as

346

the temperature increased, with the minimum at 4ºC, and the swelling degree decreased slightly

347

thereafter at higher temperatures (50 and 80ºC).

348

Incubation temperature had no effect on the major chemical groups of the stripping films

349

(Figure S8 E). The thermal stability of the stripping film prepared at different temperatures was

350

evaluated by TGA, as shown in Figure S8 F and G. All the films had a weight loss of about 13% in

351

the range of 45-120ºC, owning to the release of water molecules from the films. The films made at

352

25ºC and 80ºC had maximum weight loss at 237.3 and 238.1ºC, while those made at 4, 10, and


Page 16 of 37

Page 17 of 37


353

50ºC had maximum weight loss at 219.7, 220.9, and 245.7ºC, respectively.

354

Assembling layer numbers of films. The (chitosan/sodium phytate)1 film (first layer) could

355

not form perfectly, so we started from the second layer. It was determined that the increase of the

356

thickness of the films was proportional to the increase in the number of layers (Figure S9A). The

357

(chitosan/sodium phytate)2 films had a thickness of 49±1 µm, while the (chitosan/sodium

358

phytate)11 counterpart had a thickness of 113±4 µm, suggesting that the film formation process

359

was uniform and repeatable. At room temperature, we tested the mechanical properties of films

360

with different numbers of layers (Figure S9B). We found that the tensile properties gradually

361

enhanced with the increasing number of layers and (chitosan/sodium phytate)10 films, with a

362

maximum stress of 38.81 MPa, and also had a uniaxial stretchability of 12.7%, which is superior

363

to other films. Hence, we chose chitosan/sodium phytate)10 films for the subsequent study. The

364

resultant (chitosan/sodium phytate)10 films exhibited much higher tensile strength (MPa) than the

365

chitosan and sodium alginate LBL thin films (13.0 MPa) reported by Mandapalli et al. 34

366

The surface morphology and fractured cross-section structure of films were confirmed by

367

SEM, as shown in Figure S9C-V. It is clear that the LBL films had a smooth surface. The FTIR

368

spectra of LBL casting films presented strong absorption at 3000-3500 cm-1, related to the

369

stretching vibration of the hydroxyl groups (O-H), with an increasing number of layers (Figure

370

S10), which indicated that the interactions between chitosan and sodium phytate became stronger

371

with the increasing number of layers.

372

Moisture content, glass transition temperatures, and TGA. The moisture content of the

373

stripping and casting films increased as the RH increased (Figure S11A). The highest moisture

374

content was 36.84 ± 0.37 wt% for casting film under an RH of 97%. It appeared that dried films



375

still had a negligible amount of water. The films were dried in a vacuum oven at 40°C for 24 h;

376

however, bound water molecules still existed. Previously, Agarwal et al. 35 reported that water

377

molecules strongly bound to silks in the films of regenerated Bombyx mori silk. To study the glass

378

transition in both stripping and casting films, DSC experiments were performed (Figure S11B, C).

379

The glass transition temperature of stripping and casting films were detected between -35 and

380

-40°C. The results showed that the glass transition temperature of the films was hardly dependent

381

on the moisture content of the films. The possible reason was that the WVP values of films were

382

four to five orders of magnitude lower than other chitosan-based films, so the films we prepared

383

had low water absorption. Moreover, the DSC results of films showed no endotherm peak of ice

384

melting, indicating that water in the films were bound water rather than free water. Therefore,

385

different humidity did not affect glass transition temperature of films.

386

Both stripping and casting films incubated at different RHs were examined by TGA, and a

387

three-step weight decrease was detected (Figure S11D and G). The first weight decrease, which

388

was due to the water removal (evaporation), proceeded up to approximately 200°C and was

389

dependent on the RH. The second decrease in weight, which was attributed to thermal degradation,

390

was detected above 250°C, when the film turned into black char. The derivative plot showed the

391

temperature at which the water removal and degradation occurred at the fastest rates (Figure S11E,

392

F, H and I). The degradation temperature was independent of the water content, suggesting that the

393

water molecules were completely removed at 200°C, and the breakage of the primary chain in the

394

chitosan molecules occurred without water. Thus, the thermal degradation of the films occurred

395

independent of the moisture content. Conversely, water removal was dependent on the moisture

396

content of the films.


Page 18 of 37

Page 19 of 37


397

Stability of films. The erosion stability of the films was evaluated in buffer media with

398

different pH values, ranging from 2 to 9 at 25ºC (Figure S12). The stripping films displayed

399

excellent stability over the whole pH range. The erosion values increased from 6.7±0.3% to 30.2 ±

400

0.7%, with the pH increasing from 2 to 9. It would be expected that the films started to slightly

401

erode above pH 6 (16.7±0.3%). When the stripping films were immersed in an alkaline

402

environment (pH >7), the films still maintained their structural integrity, even up to pH 9 (Figure

403

S12 A). However, a significant increase in the erosion of casting films was observed at pH 6

404

(25.8± 0.03%) relative to the other acidic pHs (Figure S12 B). The erosion values increased

405

substantially, ranging from 26.7 ± 0.04% (pH 7) to 72.1 ± 1.1% (pH 9) (Figure S12 B). Altogether,

406

under all the conditions, the erosion values of the stripping films were lower than that of the

407

casting films, showing preferable pH stability compared to other natural polyelectrolytes films.36

408

The erosion values of both stripping and casting films increased with the increasing of

409

temperature from 4 to 60ºC (Figure S13) as a result of the destruction of a network structure of

410

films. Interestingly, both stripping and casting films retained their temperature response as

411

chitosan chains were dissociated from the network. Compared to casting films, stripping films had

412

higher thermal stability. This result was in accordance with the chitosan-based films reported by

413

Leceta et al. 37

414

Interaction mechanism. The X-ray diffraction pattern of pure chitosan, sodium phytate, and

415

chitosan-sodium phytate films are shown in Figure S14A and B. The XRD pattern of pure chitosan

416

presented two characteristic peaks at 2θ = 10.6º and 2θ = 20.1º, which are related to the

417

occurrence of hydrated hydrogen-bonded chitosan crystallized phase form II. 38 However, both the

418

stripping and casting films revealed a very poor diffraction pattern, illustrating the amorphous



419

phase of the films. Significantly, there was a peak in casting films with an intensity maximum

420

around 9.3º, indicating that the casting films contained extra chitosan chains. 39

421

The FTIR spectra of pure chitosan, sodium phytate, and composite films are shown in Figure

422

S14 C and D and Figure S15. Regarding chitosan, the strong absorption at 3346 cm-1 is related to

423

the stretching vibration of the hydroxyl groups (O-H), as well as intermolecular hydrogen bonding

424

within the polysaccharide. The characteristic absorption peak appearing at 2866 cm-1

425

corresponded to the C-H stretching vibration. Two characteristic bands at 1633 cm-1 and 1540 cm-1

426

are due to C-O stretching (amide I) and N-H bending (amide II), respectively. The characteristic

427

bands of sodium phytate are centered at 965, 1074, and 3147 cm-1. The bands of 965 cm-1

428

corresponded to PO43- and the 1074 cm-1 is the characteristic band of P=O, while the peak at 3147

429

cm-1 is assigned as the OH-characteristic band.40 After film formation, the stripping film presented

430

the characteristic peak of chitosan, but the peak of chitosan disappeared at 2866 cm-1. The

431

hydroxyl groups and amide II band of the casting films shifted from 3346 to 3267 cm-1 and 1540

432

to 1547 cm-1, respectively. Furthermore, the new peak at 1389 cm-1 appeared due to the

433

intermolecular interactions between chitosan and sodium phytate, which clearly confirmed the

434

electrostatic interactions between the phosphoric groups in sodium phytate and the amino groups

435

in chitosan.

436

To further analyze the interactions involved in chitosan-sodium phytate films, the

437

dissociation test of films in dissociation reagents was studied. The dissociation reagents used were

438

NaCl and urea, which can disrupt electrostatic interactions and hydrogen bonds, respectively. The

439

erosion of stripping and casting films remarkably increased with increased concentrations of NaCl

440

and urea (Figure 6), indicating that the formation of films occurred through both electrostatic


Page 20 of 37

Page 21 of 37


441

interactions and hydrogen bonds. It was found that the ionic strength had a more noticeable effect

442

on the erosion of casting films than that of stripping films, while the stripping films mainly took

443

advantage of hydrogen bonding attractions between chitosan and sodium phytate. The strong

444

electrostatic interactions between the amidogen groups of chitosan and the phosphate groups of

445

sodium phytate would significantly densify the film structure and subsequently enhance its

446

mechanical properties.

447

Figure 6C represents the interplay change between the chitosan chains and sodium phytate of

448

films, as well as their stability, across the entire pH range. In acidic media, chitosan is fully

449

charged, and the films presented lower dissipation due to the combination of the ionization of

450

phosphate and amine groups, which revealed that the films became more rigid. A similar behavior

451

was already reported for gelatin-poly (galacturonic acid). 41 At pH 8, the

462

electrostatic repulsions between the negative charges of sodium phytate increased (drastic increase



463

in ζ potential) and the structural integrity for casting film was not maintained. This could be due to

464

the formation of casting film mainly through electrostatic interactions, resulting in a largely partial

465

dissolution of the films under alkaline conditions. The stripping film mainly depended on

466

hydrogen bonding, leading to only a partial dissolution of the film.

467

Antibacterial activity. Figure 7 (A) and (B) show representative images of E. Coli incubated

468

with the samples for 4 h for stripping and casting film, respectively. The pure chitosan and sodium

469

phytate solution presented a high quantity of living bacteria. For stripping films prepared with 3.0

470

wt% chitosan, there was no living bacteria when the concentration of sodium phytate was

471

increased to 2.5 wt%. With regard to casting films with 4.0 wt% chitosan, the bacteria were fully

472

inhibited above 1.25 wt% concentration of sodium phytate. Meanwhile, the number of colonies on

473

the agar was statistically analyzed. As shown in Figure 7C and Table S5, when the concentration

474

of sodium phytate was above 2.0 wt%, the stripping films and casting films exhibited excellent

475

antibacterial activity against S. aureus and E. coli (nearly 100%). Our results suggested that

476

chitosan-sodium phytate films prepared by casting technologies were superior to film-stripping

477

technologies in terms of antibacterial activity. This was due to higher solubility of casting films

478

than that of stripping films (Figure 3 and S3), which could lead to the release of higher levels of

479

antimicrobial substances.

480

In summary, we first prepared chitosan-sodium phytate films using one-step consecutive

481

stripping and layer-by-layer casting methods. The resultant films showed good water vapor

482

resistance properties. Moreover, their shapes were maintained even after 1 h of soaking in acidic

483

conditions. The yellowish color of the casting films indicated the strong electrostatic interactions

484

between chitosan and sodium phytate, which was supported by the FTIR spectra. The chitosan


Page 22 of 37

Page 23 of 37


485

films containing sodium phytate could improve UV barrier properties. No classical plasticizing

486

effect of the water on the glass transition was evident in the case of films. Importantly,

487

antibacterial experiments indicated that these casting films exhibited extremely efficient

488

antibacterial activities, indicating that chitosan-sodium phytate films can serve as a promising

489

coating to inactivate microorganisms. The chitosan-sodium phytate films provide a platform for

490

the engineering and assembly of advanced materials for food, medical, and industrial uses.

491

Supporting Information Available:

492

UV-vis transmission, opacity, WVP, solubility, swelling degree, moisture content, DSC, TGA,

493

Tg, XRD, mechanical properties and antibacterial rate of the films fabricated under other

494

preparation conditions; SEM analysis of casting films.

495

Notes

496 497 498

The authors declare no competing financial interest. ACKNOWLEDGMENTS

This work was supported by the Special Funds for Taishan Scholars Project of Shandong

499

Province (No. ts201712058).

500

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Page 28 of 37

Page 29 of 37


Table 1. The thickness, opacity and WVP of the filmsa. Concentration of sodium phytate (%)b

Thickness (µm)c

Opacity

4.556±0.220a 4.300±0.141ab 4.083±0.160b 3.059±0.143c 2.700±0.113d 2.308±0.102e 1.214±0.051b 0.992±0.011d 1.048±0.032cd 1.141±0.101bc 1.195±0.049b 1.492±0.053a

Stripping film technique

0.625 1.25 2.0 2.5 3.0 4.0

9±1e 10±4de 12±3de 17±3cd 20±4bc 26±3ab

Casting film technique

0.625 1.25 2.0 2.5 3.0 4.0

70±3d 85±4c 105±3b 124±6a 128±4a 130±6a

a

WVP (×10-11 g/m×h×Pa) 2.902±0.131c 1.654±0.062e 1.786±0.076e 2.109±0.101d 2.811±0.128c 3.440±0.099b 4.052±0.182e 6.326±0.262d 6.946±0.262d 8.203±0.364c 11.642±0.541b 16.124±0.801a

The values correspond to the mean ± standard deviation. Values within each column followed by different letters indicate significant differences (P < 0.05). b The concentrations of chitosan were 3.0% and 4.0 wt% for the stripping and casting films, respectively. cThe thickness values for the casting films correspond to ten layers.



Chitosan

Page 30 of 37

Chitosan solution

Dissolution in acetic acid Stand degassing Stripping-film technique

Layer-by-layer casting technique

Sodium phytate Chitosan casting

Repeat

Repeat

Distributed on the surface of chitosan

Evenly coated Stripping-film

Film wash in water 45ºC drying

Air drying

Repeat for two times

Two layers of film

Repeat for ten times

Ten layers of film

Film

Soft, transparent

Ultrasoft, supertransparent

Figure 1. Preparation chart for chitosan-sodium phytate film using the film-stripping and layer-by-layer casting technique.


Page 31 of 37


0.625% 1.25% 2.0% 2.5% 3.0% 4.0%

Transmittance (%)

100 95 90

0.625% 1.25% 2.0% 2.5% 3.0% 4.0%

B 100 Transmittance (%)

A 105

85 80 75

80

60

40

70 65 60 260

20

280

300

320

340

360

260

280

300

320

340

360

Wavelength (nm)

Wavelength (nm)

C

D

E

F

Figure 2. (A) and (B) Light transmittance results at various concentrations of sodium phytate; (C) and (D) The photograph of the films and the concentration of sodium phytate from left to right: 0.625, 1.25, 2.0, 2.5, 3.0, and 4.0 wt%; (E) and (F) Digital photographs showing the flexibility of the films with 1.25 wt% of sodium phytate. (A), (C), and (E) The film prepared by film-stripping technique and the concentration of chitosan at 3.0 wt%; (B), (D), and (F) The film prepared by LBL technique and the concentration of chitosan at 4.0 wt%.



ab

80

bc

c

20

d

d

70 60

a b

b

b

b

B 3000

b

b

a

b c

P ercentage (% )

90

a

Solubility Swelling degree Moisture content

2000

40

c

30

c

b

c

b

c ab

a

b b

b

20

a

a

0

1.25

2.0

2.5

3.0

0.625

4.0

Concentration of sodium phytate (%)

E

80 70

0

5

10

15 20 Strain (%)

25

0.0000

-1

90

Deriv. mass (wt% °C )

0.625% 1.25% 2.0% 2.5% 3.0% 4.0%

100

20

0

1.25 2.0 2.5 3.0 4.0 Concentration of sodium phytate (%)

D 110 W e ig h t (% )

60 50

-0.0002 -0.0004

0.625% 1.25% 2.0% 2.5% 3.0% 4.0%

-0.0006 -0.0008 -0.0010

40

-0.0012

30 200

300

400

500

600

Temperature (°C)

F 100

0.625% 1.25% 2.0% 2.5% 3.0% 4.0%

90 80 70 60

100

G

0.0002 0.0000

-1

100

Deriv. mass (wt% °C )

0.625

30

10

10

0

0.625% 1.25% 2.0% 2.5% 3.0% 4.0%

C 50

d

d

1000

d

10

a

a

b

Stress (MPa)


a

W eig ht (% )

Percentage (% )

A

Page 32 of 37

200 300 400 Temperature (°C)

500

600

-0.0002 0.625% 1.25% 2.0% 2.5% 3.0% 4.0%

-0.0004 -0.0006 -0.0008 -0.0010 -0.0012

50

-0.0014 100

200

300

400

500

Temperature (°C)

600

-0.0016 100

200

300

400

500

600

Temperature (°C)

Figure 3. The solubility, swelling degree, and moisture content of the stripping film containing 3.0 wt% of chitosan (A), LBL film containing 4.0 wt% of chitosan (B); (C) Stress-strain curves of casting films with different concentrations of sodium phytate incubated at RH 75%. The concentration of chitosan is 4.0 wt%. TGA curves of films prepared by stripping film containing 3.0 wt% of chitosan (D, E) and LBL film containing 4.0 wt% of chitosan (F, G) with different concentrations of sodium phytate. Data are presented as the average of triplicate measurements with standard deviation (n = 3). Values with different letters (a, b, c, and d) are significantly different (p < 0.05).


30

35

Page 33 of 37


A

5s

0s

60s

140

C 120

120

110

Transmittance (%)

Thickness (µm)

B

30s

15s

100 80 60

90s

2min

3min 1min 5min 10min 20min 30min

100 90 80 70

40

60

20

50 40

0 0

5

10

15

20

25

260

30

280

Formation time (min) 1min 5min 10min 20min 30min

100 90

E

340

360

0.0002 0.0000

D eriv. m ass (w t% ° C -1 )

W eight (% )

D

300 320 Wavelength (nm)

80 70 60

-0.0002

1min 5min 10min 20min 30min

-0.0004 -0.0006 -0.0008 -0.0010 -0.0012

50 100

200

300

400

500

600

-0.0014 100

Temperature (°C)

200

300

400

500

Temperature (°C)

Figure 4. (A) Chitosan/sodium phytate film formation time observed from the solution flowing in the inverted vials after different time. Five milliliter 3.0 wt% chitosan was mixed with 1ml 1.25 wt% sodium phytate. (B) Thicknesses of film as a function of film-forming time. (C) UV-vis transmission spectra of the films prepared by film-stripping technique at various formation time. The concentration of chitosan and sodium phytate are 3.0 wt% and 1.25 wt%, respectively. (D) Thermal degradation of stripping films with different time. Weight changes in the film. (E) Derivative plot of (D).


600


1 2 3 4 5 6 7 8 9 10

Transm ittance (% )

100 95 90


B ab

90 Percentage (%)

A 105

Page 34 of 37

ab

ab

ab

ab c

abc

ab

a

bc

80 70 20

bc a

ab

a

a a c a

a

ab

a

a bc a

a

a

ac a

a

85

10 80 260

280

300

320

340

360

0 1

2

1

2

10 µm

10 µm

6

10 µm

D

1

50 µm

7

50 µm

10 µm

8

10 µm

2

3

50 µm

50 µm

50 µm

5

6

7

8

9

10

11

4

5

10 µm

10 µm

3

10 µm

7

4

Stripping film Number

Wavelength (nm)

C

3

8

50 µm

9

10 µm

4

50 µm

9

50 µm

Figure 5. (A) UV-vis transmission spectra, (B) The solubility, swelling degree, and moisture content of the film, (C) Surface morphology. (D) Fractured cross-section structure of stripping films prepared at different stripping film numbers. Number 1 to 10 represents films formed at the first stripping to tenth stripping, respectively. Data are presented as the average of triplicate measurements with standard deviation (n = 3). Values with different letters (a, b, and c) are significantly different (p < 0.05).


10

10 µm

5

50 µm

10

50 µm

Page 35 of 37


B 100

A 100

Stripping film Casting film

Erosion (% )

Erosion (%)

Stripping film Casting film 80

60

80

60

40

40 20

20 0

10

50

100

250

1

500

2

C OH

3

4

OH

H PO H3PO4 3 4OH

PO43- PO 34 OH

OH

2

NH3+

P =O

HO

H3PO4 NH +H3PO4 3 OH OH NH3+

PO43-PO

HO HO

OH

OH NH2

H2N

NH3+ NH2

3

4 OH

5 PO43-

3PO43- PO4

OH

NH3+

6

7

OH

pH 8

9

PO43-

PO43OH

PO43- PO43PO 3PO43- 4

OH

PO43PO43NH3+

NH2

NH2 NH3

+

H Hydrogen interaction

PO43-

3-

4

OH

Sodium phytate

5

Urea concentration (M)

Salt concentration (mM)

Chitosan

N H

HO

NH2 OH

OH

Electrostatic interaction

Figure 6. The influence of NaCl (A) and urea (B) on the erosion of the chitosan-sodium phytate films. (C) Illustration that shows the effect of pH on the molecular mobility of films and on the charges and interactions behind chitosan-sodium phytate films.



E. Coli. only

Page 36 of 37

1.25% Sodium phytate

4.0% Chitosan

A

3.0%+0.625%

3.0%+1.25%

3.0%+2.0%

3.0%+2.5%

3.0%+3.0%

4.0%+0.625%

4.0%+1.25%

4.0%+2.0%

4.0%+2.5%

4.0%+3.0%

B

Stripping films Casting films

Antibacterial ratio (%)

C 100

a

a

b

80

a

a

a a

2.5

3.0

b

60 40 20 0

c

c

0.5

1.0

1.5

2.0

Sodium phytate (%) Figure 7. (A) and (B) Quantitative calculation of E. Coli cultivated on LB agar covered with bacteria suspension that was in contact with stripping films (3.0 wt% chitosan) and casting films (4.0 wt% chitosan) with different concentrations of sodium phytate, respectively. (C) Antibacterial ratio of chitosan-sodium phytate film against E. Coli. Data are presented as the average of triplicate measurements with standard deviation (n = 3). Values with different letters (a, b, and c) are significantly different (p < 0.05).


Page 37 of 37


For Table of Contents Only

Stripping film

Stripping film

Casting film

Casting film


Chitosan-sodium phytate films with a strong water barrier and

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