chitosan composite film for potential antimicrobial applications

Journal Pre-proof Preparation and characterization of pullulan derivative/chitosan composite film for potential antimicrobial applications

Shubin Li, Juanjuan Yi, Xuemei Yu, Zhenyu Wang, Lu Wang PII:

S0141-8130(19)35502-3

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.01.080

Reference:

BIOMAC 14384

To appear in:

International Journal of Biological Macromolecules

Received date:

16 July 2019

Revised date:

25 December 2019

Accepted date:

8 January 2020

Please cite this article as: S. Li, J. Yi, X. Yu, et al., Preparation and characterization of pullulan derivative/chitosan composite film for potential antimicrobial applications, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/ j.ijbiomac.2020.01.080

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2018 Published by Elsevier.

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Preparation

and

characterization

derivative/chitosan

composite

film

of

pullulan

for

potential

antimicrobial applicatihtns Shubin Li a, Juanjuan Yi b, Xuemei Yu a, Zhenyu Wang a, Lu Wang a* a

Harbin Institute of Technology, 92 Xidazhi Road, Nangang District, Harbin 150001,

School of Life Sciences, Zhengzhou University, Zhengzhou 450001, PR China.

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b

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PR China

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*Corresponding author: Lu Wang

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Postal address: School of Chemical Engineering and Chemistry, Harbin Institute of

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Technology, 92 Xidazhi Road, Nangang District, Harbin, 150001, PR China. E-mail

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Address: [email protected]

Tell numbers: 0451-86282909, Fax numbers: 0451-86282909.

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Nonstandard Abbreviations: Adipic acid dihydrazide: ADH; Oxidized pullulan: OxPullulan; Thermogravimetric analysis: TGA; Scanning electron microscopy: SEM; tensile strength: TS; Elongation-at-break: EB; Relative humidity: RH; Water vapor transmission rate: WVP;

ABSTRACT: Antimicrobial food-packaging films can improve the safety of food and prolong shelf life. In the present work, we first prepared the pullulan derivative and then added chitosan to form a pullulan derivative/chitosan composite film. The active amino

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groups on the pullulan derivative backbone and chitosan impart antimicrobial activity against S. aureus. Also, the addition of chitosan made the composite film show reasonable mechanical properties, when the ratio of pullulan derivative:chitosan=2:8 and the tensile strength was 21 MPa. The composite film also behaved with the excellent film-forming property, Water vapor barrier, and light barrier properties. This

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work is opening the door on preparing a novel antibacterial packaging film.

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KEYWORDS: Pullulan; Chitosan; Antibacterial; Composite film; Food-packaging

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material

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1. Introduction

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With the continuous increases of people's needs for food safety, the food packaging field has significantly been developed [1-6]. In the past few decades, polymers (like

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plastic) have been widely used in food packaging due to the reason that they have

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many advantages. For instance, they are low cost, light weight, heat sealability and they can also change the composition and shape of the package according to different demands [7-10]. However, these polymers are tough to degrade in nature, and a large part of the waste will spread, migrate and accumulate in the environment. This will extremely harmful to humans and other living things [11, 12]. The most effective way to solve this problem is to use naturally degradable materials. Among them, natural polysaccharides have attracted great attention due to their high film-forming properties, functional nutritional properties, and chemical stability [13-15]. As a common natural microbial polysaccharide, pullulan is an ideal raw material for the preparation of food packaging materials due to its excellent biodegradability,

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safety, and thermal stability [16, 17]. However, natural pullulan does not have any antibacterial ability, which is a crucial factor limiting its widespread use [18]. To solve this problem, many studies have focused on enhancing the antimicrobial properties of pullulan. Adding antibacterial agents to the film, such as nanoparticles [19-21] and essential oils [22, 23] can improve the antibacterial ability of the film, but

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these antibacterial agents usually require complicated preparation or extraction processes, which significantly increase the difficulty in preparation of the film.

the

antimicrobial

capacity

of

the

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enhance

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Therefore the chemical modification of pullulan has become the preferred method to film.

For

example,

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3-aminopropyltrimethoxysilane was used to prepare pullulan derivative to prepare

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antibacterial transparent pullulan film, which improved the thermal stability and

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antibacterial ability of the film [24].

However, a single component film generally does not meet the packaging material's

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requirements for the mechanical properties of the film. It is usually necessary to add additional film-forming substances for improvement. For example, the incorporation of chitosan in polyethylene oxide (PEO) films can improve mechanical properties and contribute to the formation of films [25]. Here, we prepared a pullulan derivative and added chitosan to prepare a pullulan derivative/chitosan composite film. The active amino group was introduced by using adipic acid dihydrazide (ADH) modified pullulan to improve the antibacterial property of pullulan. In addition, due to the good antibacterial property and film forming property, chitosan was added to the film to further improve the antibacterial

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and mechanical properties. This study provides a theoretical basis for the preparation of new degradable antibacterial composite film and the promotion of the application of pullulan in food packaging.

2. Materials and methods 2.1. Materials

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Pullulan (average molecular weight [MW] of approximately 800,000) was

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purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China), ADH, sodium

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periodate and sodium cyanoborohydride were obtained from Sigma Aldrich

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(Shanghai, China). Chitosan (CS) (average MW of approximately 100000) was purchased from Aldrich Chemical Company (St Louis, USA). The dialysis bags

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(molecular weight cutoff 8.0-12.0 kDa) purchased from Solarbio Science ﹠

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Technology Co., Ltd. (Beijing, China)

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2.2. Preparation of Pullulan derivative 2.2.1. Preparation of oxidized pullulan (OxPullulan) The oxidation of pullulan was carried out as per procedure reported in the literature [26]. Briefly, 1 g of pullulan (0.006 mol) and 0.66 g of sodium periodate (0.003 mol) were dissolved in 30 mL of distilled water. The solution was stirred in the dark at 25 °C for 5 h. After that, the solution was purified by dialysis against distilled water for 48 h. Finally, the solution was freeze-dried to obtain the OxPullulan. The contents of aldehyde group in OxPullulan were determined by using hydroxylamine hydrochloride method [27].

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2.2.2. Preparation of adipic acid dihydrazide-oxidized pullulan (ADH-OxPullulan) OxPullulan (0.1 g) in 30 mL of distilled water was mixed with ADH (1.74 mg, 0.01mmol). The solution was stirred under magnetic stirring for 4 h. Then, 0.64 mg NaCNBH4 (0.01 mmol) was added to the mixture and stirred for 24 h at room

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temperature. To eliminate any unreacted components, the samples were dialyzed

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thoroughly with distilled water. The ADH-OxPullulan was obtained by using

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freeze-dried.

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2.2.3. Preparation of ADH-OxPululan/Chitosan films

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ADH-OxPullulan and CS were respectively weighed at mass ratios of 10:0, 8:2, 6:4, 4:6, 2:8 and 0:10, then dissolved in 1% acetic acid aqueous solution (v/v) for 30

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min. The composite film solution was poured into a Teflon-coated dish (6 cm×10

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cm). The solution was dried at 50 °C until the solvent was evaporated completely. All the samples were stored in 50%±3% relative humidity (RH) at room temperature.

2.4. Characterization of synthetic substances 2.4.1. Fourier transform infrared spectroscopy (FTIR) The chemical structure of the samples was determined by FTIR spectrometer (Perkin Elmer Instruments Ltd, USA). The spectra were collected in the range from 500 to 4000 cm-1.

2.4.2. Nuclear magnetic resonance spectroscopy (NMR)

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The

structure

of

samples

was

further

confirmed

by

using

1

H-NMR

spectrophotometer (Bruker Avance III 400, USA). The sample was solubilized in DMSO/D2O (0.5 N) and tested at room temperature. The degree of substitution of ADH was calculated by the ratio of methylene protons to sugar protons with the following equation [28]: 100%

Area(5.32 ppm)

(2)

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Substitution degree of ADH=

1 ×[Area(1.74 ppm)+Area(2.41 ppm)] 8

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2.4.3. X-ray diffraction (XRD)

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X-ray diffraction (XRD) patterns were recorded using an XRD-diffractometer (PANalytical B.V., Netherlands). The scan range was from 5° to 90°.

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2.4.4. Thermogravimetric analysis (TGA)

Thermogravimetric analysis (TGA) was carried out with a TG/DAT 6300 (Hitachi,

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Japan) with a heating rate of 10 °C/min under nitrogen atmosphere.

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2.4.5. Gel permeation chromatography (GPC) The molecular weight of samples was obtained by using a Shimadzu-LC2010C gel permeation chromatography (Shimadzu Manufacturing Co., Ltd., Japan). Preparing 1% sodium azide standard solution as mobile phase, and the flow rate was 1 mL/min.

2.5. The properties of composite films 2.5.1. Films thickness The thickness of films was determined by a digital micrometer caliper (0-25 mm, Mitutoyo, Mitutoyo Corporation, Japan). The accuracy of caliper was 0.001 mm. Each sample was measured ten random points and reported the average value.

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2.5.2. Water and Light properties of composite films The water vapor transmission rate (WVP) of the films was measured by using the normative testing method of active standard ASTM E96/E96M [29]. Briefly, the anhydrous calcium chloride was put anhydrous calcium chloride into the beaker, then sealed the beaker with a film (d=10 mm). The beaker was placed in a container, which

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contained distilled water at the bottom. The beaker was weighed per hour, and WVP

Δm × d A × Δt × Δp

(1)

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WVP =

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was determined according to Eq. (1):

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Where ∆m was the quality increase, g; A was the film area, m2; ∆t was the time

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interval, s; d was the film thickness, m; ∆p was the water vapor pressure difference on both sides of the sample film, kPa.

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The 5100 ultraviolet-visible (UV-Vis) spectrophotometer (Hitachi, Japan) was used

800 nm.

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to evaluate the light transmission of the films. The measured range was from 200 to

2.5.4. Mechanical properties The tensile strength (TS) and breaking elongation rate (EB) of specimens were tested using a texture analyzer (TA-Xtplus, Stable Micro Systems, UK). The stretching rate was 50 mm/min.

2.5.5. Morphology observation

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The morphologies of films were examined by scanning electron microscopy (SEM) (Quanta 200, FEI, USA). Photographs were taken at acceleration voltages of 5 kV electron beam.

2.5.6. Antibacterial activity assay The bactericidal activity of films was evaluated by using the agar diffusion method.

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In brief, 0.1 mL of bacteria suspensions (108 CFU/mL) of S. aureus was added into

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the agar medium. The films were cut into circular discs 6 mm in diameter and then

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were placed on the medium previously inoculated with the S. aureus. Each plate was incubated at 37 °C for 24 h. The inhibition zone was recorded in order to evaluate the

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2.6. Statistical analysis

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antibacterial effect.

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All tests were repeated at least three times. The statistical significance was

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measured using an analysis of variance (ANOVA). Significance was defined at P values of < 0.05.

3. Results and discussions 3.1. Characterization of synthetic substances Fig. 1 illustrated the preparation process of the pullulan derivative. The process was including two steps: (1) sodium periodate oxidation was used to prepare OxPullulan; (2) ADH was employed to modify the OxPullulan to obtain ADH-OxPullulan.

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Fig. 1. The synthesis scheme of pullulan derivative.

3.1.1. FTIR analysis.

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The FTIR spectra of pullulan and pullulan derivative were shown in Fig. 2(a). For pullulan, the absorption peaks at 3400 cm-1, 2930 cm-1, 1640 cm-1, 913 cm-1, and 768 cm-1 were assigned to the stretching vibration of O–H, –CH2–, O–C–O, α-1,6-glycosidic linkage and α-1,4-glycosidic linkage, respectively [30, 31]. After oxidation, OxPullulan presented a new peak at 1730 cm-1, which was attributed to the aldehyde groups [32]. The oxidation degree of OxPullulan was measured to be 40% by using the hydroxylamine hydrochloride/potentiometric titration method. For ADH-OxPullulan, the peaks at 1730 cm-1 disappeared. A new adsorption band appeared at 1051 cm-1, which was due to the C-N stretching vibration [33]. Moreover,

Journal Pre-proof the N-H stretching vibration at 1551 cm-1 and 3300 cm-1 showed that the presence of free amino group [34]. These results confirmed that pullulan derivative had been synthesized successfully.

3.1.2. 1H NMR analysis 1

H NMR was used to further confirm the successful synthesis of ADH-OxPullulan.

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As shown in Fig. 2(b), the signals at 4.46–5.56 ppm were assigned to the protons of

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the sugar skeleton of pullulan [35]. The peaks at 1.7 ppm and 2.4 ppm were

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attributable to the methylene peaks of ADH [36]. The degree of substitution calculated from the spectra for ADH in the pullulan was 15.5%.

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3.1.3. XRD analysis

XRD analysis was also used to determine the synthesis of a derivative. As shown in

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(Fig. 2(c)), the pattern of pullulan showed two peaks at 19.4° and 34.9°, which

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indicated that pullulan had a high crystalline structure [37]. ADH-OxPullulan had no characteristic crystalline peaks because the addition of small molecules on the macromolecular chain backbone of pullulan reduced the formation of crystal structure [38].

3.1.4. TG analysis The modification was usually accompanied by changes in thermal properties. Therefore, we finally determined the synthesis of the derivative by TG analysis. As can be seen from Fig. 2(d), samples had a slight mass loss (nearly 200 °C), which was due to the volatilization of water [39]. The mass loss of pullulan was from 270 °C to 340 °C, which was mainly caused by thermal degradation of the pullulan molecular

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chain [40]. The maximum mass loss temperature of pullulan was 305 °C. The mass loss of ADH-OxPullulan was from 250 °C to 310 °C, and the maximum mass loss temperature of ADH-OxPullulan was 274 °C. The results showed that the thermal property of ADH-OxPullulan was lower than pullulan. This was because the introduction of the ADH destroyed the hydrogen bonding of the pullulan [27]. The

Fig.

2.

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above results proved that ADH-OxPullulan was successfully synthesized.

Characterization

of

synthetic

substances.

(a)

FTIR

spectra

of

Pullulan/OxPullulan/ADH-OxPullulan. (b) 1H NMR of spectra of ADH-OxPullulan. (c)

XRD

patterns

of

ADH-OxPullulan.

(d)

TGA

curves

of

Pullulan/OxPullulan/ADH-OxPullulan.

3.1.5. GPC analysis As shown in Table 1, the GPC analysis was used to determine the molecular weight of pullulan and OxPullulan. The polydispersity index (PDI) was calculated as follows:

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PDI 

Mw Mn

(3)

Where Mw represents the mass-average molecular weight, Mn represents the number-average molecular weight. It was found that the molecular weight of OxPullulan was significantly lower than the pullulan. This finding was because sodium periodate oxidation led to the

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degradation of the polymer chain [41].

Pullulan

595057

OxPullulan

392525

Mn (g/mol)

PDI

482407

1.233

307375

1.277

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Mw (g/mol)

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Sample

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Table 1. The molecular weight of pullulan and OxPullulan

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3.2. FTIR analysis of composite films

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FTIR spectroscopy was used to examine the interactions between ADH-OxPullulan and CS. (Fig. 3) For the ADH-OxPullulan film and CS film’s spectrum, the broadband at 3331 cm-1 and 3359 cm-1 were attributed to the O–H stretching. This was caused by hydrogen bonds between molecules [42]. For composite film, the peaks of O–H stretching shifted to a higher frequency, indicating the formation of the hydrogen bond between the ADH-OxPullulan and CS [43].

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Fig. 3. The FTIR of ADH-OxPullulan/Chitosan films

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3.3. Film Thickness

Film thickness is an essential indicator of physical properties. The difference in

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film thickness may be due to the difference in structure compactness of the film [44].

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As displayed in Table 2, the thickness of the composite films was lower than that of the single-component films. It was because the hydrogen bond was formed between ADH-OxPullulan and chitosan in the composite film, which enhanced the compactness of the film structure. Table 2. The thickness of ADH-OxPullulan/Chitosan composite films Film composition Thickness (mm) (ADH-OxPullulan: Chitosan) 10:0

0.020±0.001

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0.016±0.002

6:4

0.013±0.004

4:6

0.010±0.003

2:8

0.013±0.004

0:10

0.022±0.002

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8:2

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3.4. Water and light properties of composite films

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For food packaging film, controlling the barrier properties of films is necessary to extend the shelf-life of food. As shown in Fig. 4(a), composite films showed lower

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WVP value than the single-component films. The WVP values increased with the

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increase of the ratio of ADH-OxPullulan in the composite film. It was because that

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ADH-OxPullulan contained a lot of –OH groups and easily form hydrogen bonds with water molecules, which made it had a strong hygroscopic property [45].

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The light transmittance of the package plays an important role in the preservation of food because light can affect the oxidation rate of lipids. As can be seen from Fig. 4(b), all-composite films showed good UV blocking properties at 200nm-400nm. It was beneficial for the preservation of food because the most effect of light on food quality was from ultraviolet light [46]. Also, we found that pure CS film can transmit 85% visible light, when the ratio of ADH-OxPullulan to CS was 8:2, the transmittance of visible light was only 4%. It meant that the higher ratio of ADH-OxPullulan, the lower the pass rate of visible light. This was because the different pass rate of visible light of the films was related to the internal structure

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developed during film drying [47]. Here, the modification reduces the solubility of the pullulan derivative, making it easier to associate to some extent during the drying process. Therefore, the higher the proportion of ADH-OxPullulan in the film, the

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higher the heterogeneity of the film, the lower the pass rate of visible light.

films;

(b)

The

light

transmission

of

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ADH-OxPullulan/Chitosan

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Fig. 4. Water and light properties of composite films. (a) The WVP of

ADH-OxPullulan/Chitosan films.

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3.5. Mechanical properties

Packaging films typically required sufficient mechanical strength and expandability to withstand external stress, maintain their integrity and barrier properties during the packaging process. Controlling the composition of film was an effective way to adjust the mechanical properties of the film. In general, a single component film does not meet the packaging material's requirements for mechanical properties, so it is usually necessary to add additional film-forming substances. Here, we mixed CS with ADH-OxPullulan for improving the mechanical properties of the composite film. It can be seen from Fig. 5; the TS increased with the incorporation of CS in composite

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films, which had the same trend as EB. When the ratio of ADH-OxPullulan to CS was 4:6, the TS value was 1.5 times that of pure ADH-OxPullulan film. The high TS values of composite films could be attributed to the formation of intermolecular

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hydrogen bonding between ADH-OxPullulan and CS.

Fig. 5. The mechanical properties of ADH-OxPullulan/Chitosan films

3.6. Morphological characterization SEM was used to investigate the relationship between the surface morphology and the composition of the film. As shown in Fig. 6, pure ADH-OxPullulan film was crinkled. It was due to the reason that the modification reduced the water solubility of polysaccharide and the fluidity of the film-forming solution. As the proportion of CS in the composite film increased, the film became smoother. This outcome was

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because CS had good water solubility and enhanced the fluidity of the film-forming

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solution.

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Fig. 6. The SEM of ADH-OxPullulan/Chitosan films. (a):ADH-OxPullulan/Chitosan

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10:0; (b):ADH-OxPullulan/Chitosan 8:2;(c):ADH-OxPullulan/Chitosan 6:4;

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(d):ADH-OxPullulan/Chitosan 4:6; (e):ADH-OxPullulan/Chitosan 2:8;

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(f):ADH-OxPullulan/Chitosan 0:10

3.7. Antibacterial activity

S. aureus, which is considered as one of the most multidrug-resistant bacteria [48]. Therefore, in order to investigate the antibacterial properties of the composite film, we used S. aureus as an experimental strain. The antibacterial ability of the ADH-OxPullulan/CS composite film against S. aureus was shown in Fig 7. The results showed that all the test composite films had the inhibition zone. This was because that the positively charged amino group in the composite film was combined with the negatively charged bacterial cell membrane, leading to leaching of intracellular components to the outer environment and causing the death of the

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bacteria. Also, we found that the films containing ADH-OxPullulan had higher antibacterial ability than pure chitosan film, which indicated that ADH-OxPullulan had better antibacterial properties than chitosan. It was due to the reason that the antimicrobial activity of materials bearing amino functionalities grows with the increase in the chain length of N-alkyl substituents [24]. Here, the pullulan derivative

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has a longer N-alkyl-substituted chain than chitosan, so that it has stronger antibacterial properties. Good antibacterial ability provides a broad prospect for the

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application of composite film.

Fig. 7. The antibacterial activities of ADH-OxPullulan/Chitosan films.

4. Conclusions In summary, we designed and prepared the ADH-OxPullulan/CS antibacterial films. Firstly, the active amino group was introduced to improve the antibacterial property of pullulan, the FTIR, 1H NMR, XRD, TG was proved the successful synthesis of ADH-OxPullulan. After that, we added CS to further improve the antibacterial and mechanical properties of the composite film. The results showed that

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the mechanical properties of the composite film was increased with the increasing of ratio of CS. The composite film had best antibacterial properties against S. aureus when the ratio of ADH-OxPullulan to CS was 4:6. Finally, the composite films also had good water vapor barrier, UV blocking properties. This study will lay a theoretical foundation for the preparation and application of antibacterial film and

Conflict of interest

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The authors have declared no conflict of interest.

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provide potential options for packaging and storage with high lipid foods.

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Acknowledgements

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The authors gratefully acknowledge the financial support of the NSFC (No. 21371042,

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21271056 and 21571044), Thirteen five national key research and development

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projects (No. 2016YFC0500307-07), Heilongjiang Province major research projects (No.GA13B202), and the NSF of Heilongjiang Province (No. B2015003, ZD2015001,

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JJ2016JQ0037, and H2015065).

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Author Statement Shubin Li: Conceptualization, Methodology, Writing-Original draft preparation, Data curation. Juanjuan Yi: Writing-Reviewing and Editing Xuemei Yu: Data curation, Investigation

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Zhenyu Wang: Supervision

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Lu Wang: Project administration

Journal Pre-proof Highlights

• Synthesis and characterization of adipic acid dihydrazide modified pullulan.

•The ADH-OxPullulan/Chitosan composite film was successfully prepared and characterized.

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•The ADH-OxPullulan/Chitosan composite film had good water vapor barrier, UV blocking properties and antibacterial properties.