Fabrication of cellulose nanowhiskers reinforced chitosan-xylan nanocomposite films with antibacterial and antioxidant activities

Carbohydrate Polymers 184 (2018) 66–73

Contents lists available at ScienceDirect

Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Fabrication of cellulose nanowhiskers reinforced chitosan-xylan nanocomposite films with antibacterial and antioxidant activities ⁎

Yuping Bao, Hao Zhang, Qian Luan, Mingming Zheng, Hu Tang , Fenghong Huang

T ⁎

Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Oil crops and Lipids Process Technology National & Local Joint Engineering Laboratory, Key Laboratory of Oilseeds processing, Ministry of Agriculture, Hubei Key Laboratory of Lipid Chemistry and Nutrition, Wuhan 430062, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Chitosan Xylan Cellulose nanowhiskers Antibacterial Antioxidant

Antibacterial and antioxidant chitosan-xylan/cellulose nanowhiskers (CNW) nanocomposite films were successfully prepared using CNW as nanofillers. The structure and morphology of the nanocomposite films were investigated by Fourier transform infrared spectroscopy (FTIR), X-ray diffractometry (XRD), and scanning electron microscopy (SEM). The optical transmittance, thermal stability, mechanical property, and swelling property of the nanocomposite films were also evaluated. These results revealed the microstructure of the films and confirmed the good miscibility between chitosan-xylan and CNW. The improvements of tensile strength and elongation at break of the nanocomposite films confirmed the reinforcement effects of CNW. Moreover, the inhibitory effects against S. aureus and E. coli and the ABTS+ scavenging activity indicated antibacterial and antioxidant functions of the nanocomposite films. In this work, the prepared chitosan-xylan/CNW nanocomposite films, combined the antibacterial property of chitosan, the antioxidant property of xylan, and good mechanical property of CNW, could be potentially applied in food and health-related areas.

1. Introduction With human consciousness of environmental protection strengthening and concern for infectious diseases prevention increasing, ecofriendly antibacterial and antioxidant materials have aroused extensive research interests due to their pivotal role in human health and natural environment (Carocho & Ferreira, 2013; Kenny, Smyth, Walsh, Kelleher, Hewage, & Brunton, 2014). Natural biopolymers such as chitin, cellulose, and hemicellulose, etc. are ideal candidates for designing functional materials with antibacterial and antioxidant properties because of their biocompatibility, biodegradability, and special bioactivity (Aider, 2010; Azizi Samir, Alloin, & Dufresne, 2005; Ying, Jing, Feng, Zhang, & Sun, 2014). Chitosan, the deacetylated derivative of chitin, is a homopolymer of N-acetyl-D-glucosamine (GlcNAc) residues linked by β-(1–4) glycosidic bonds. Chitosan has been used in various fields such as food (Devlieghere, Vermeulen, & Debevere, 2004; Dotto, Pinto, Hachicha, & Knani, 2015; Hajji et al., 2017), pharmaceutical (Pal, Behera, Roy, Ray, & Thakur, 2013; Prabaharan, 2015), agriculture (Ngah, Teong, & Hanafiah, 2011; Zeng, Wu, & Kennedy, 2008) for its nontoxic, antibacterial, and biodegradable properties. Xylan polysaccharides, the main hemicellulose components of cell walls, consists of xylose linked by β-1,4-glycosidic bonds with 4-O-methylglucosidic acid groups

⁎

branched via α-1,2-glycosidic bonds (Ebringerova & Heinze, 2000). Xylan-based biomaterials have been widely used in food coatings (Mikkonen & Tenkanen, 2012), biosorbent (Ayoub, Venditti, Pawlak, Salam, & Hubbe, 2013), and drug carrier (Silva, Habibi, Colodette, & Lucia, 2011), due to its low cost and easy availability. Chitosan-xylan compounds formed from the interaction between the free amino groups of chitosan and the aldehyde groups of xylan chains are potential food preservations for they possess antibacterial and antioxidant activities (Li, Shi, Wang, & Du, 2011). However, the low film-forming property of chitoan-xylan compound has limited its application. In recent years, nanosized reinforcements have been widely used to develop nanocomposites with improved mechanical properties (Njuguna, Pielichowski, & Desai, 2010). Cellulose nanowhiskers (CNW), possess attractive features such as nontoxicity, biodegradability, nanosize, and high aspect ratio (Eichhorn, 2011; Martínez-Sanz, Vicente, Gontard, Lopez-Rubio, & Lagaron, 2015), have been studied as reinforcements in various natural and synthetic polymer matrices to prepare nanocomposite films with enhanced properties (Liu, Dong, Bhattacharyya, & Sui, 2017; Martínez-Sanz, Lopez-Rubio, & Lagaron, 2013; Qi, Cai, Zhang, & Kuga, 2009). The tensile strength of nanocrystalline cellulose/chitosan composite films could be raised from 33.7 Mpa to 57.2 Mpa with nanocrystalline cellulose/chitosan ratio of 30/70 (Rubentheren, Ward, Chee, & Nair, 2015). The storage modulus

Corresponding authors. E-mail addresses: [email protected] (H. Tang), [email protected] (F. Huang).

https://doi.org/10.1016/j.carbpol.2017.12.051 Received 20 August 2017; Received in revised form 13 December 2017; Accepted 19 December 2017 Available online 23 December 2017 0144-8617/ © 2017 Elsevier Ltd. All rights reserved.

Carbohydrate Polymers 184 (2018) 66–73

Y. Bao et al.

evaporated. Finally, the films were kept in a vacuum oven at room temperature to remove the trace solvent. The films were coded as CX, CX/CNW4, CX/CNW8, CX/CNW12 and CX/CNW16 according to the weight ratios of CNW to chitosan calculated to be 0, 4%, 8%, 12% and 16%, respectively.

of electrospun cellulose acetate fibers could be improved from 81 to 825 MPa with 1 wt% CNW addition, showing a significant positive effect on mechanical properties (Herrera, Mathew, Wang, & Oksman, 2011). Therefore, CNW could be introduced into polymeric matrices as nanofillers for designing functional film materials with enhanced properties. In this work, we aimed to prepare chitosan-xylan/CNW nanocomposite films using CNW as nanofillers. The structure and morphology of the nanocomposite films were investigated by FTIR, XRD, and SEM. Moreover, optical property, mechanical property, swelling property, thermal stability, antibacterial activity and antioxidant activity of these nanocomposite films were also investigated.

2.4. Characterization Transmission electron microscopy (TEM) images were observed on a JEOL JEM-2010 (HT) electron microscope at an accelerating voltage of 200 kV. Fourier-transform infrared (FT-IR) was performed on a FT-IR spectrometer (Nicolet170-SX, Thermo Nicolet Ltd., USA) in the wavenumber range of 4000–400 cm−1. Wide angel X-ray diffraction (WAXD) analysis was recorded on a WAXD diffractometer (MiniFlex 600, Rigaku, Japan) with a scanning scope of 5–40° and a scanning speed of 5°/min−1, using Cu Kα radiation at 40 kV and 15 mA. The crystallinity of the native cellulose and CNW was calculated using the following equation (Gümüskaya, Usta, & Kirci, 2003; Sunkyu, Baker, Himmel, Parilla, & Johnson, 2010):

2. Experimental 2.1. Materials Commercial grade chitosan from shrimp shell was purchased from Ruji Biotechnology Co. Ltd. (Shanghai, China) with a deacetylation degree of 89%. Its weight-average molecular weight measured by dynamic light scattering (DLS) in LiOH–KOH–urea aqueous solution was 36.0 × 104 Da (Duan, Liang, Cao, Wang, & Zhang, 2015). Xylan from corn cobs was purchased from Hanhong Ltd. (Shanghai, China) with a molecular weight of 3.24 kDa measured by DLS (Li, Shi, Wang, & Du, 2011). Cellulose from cotton linter pulp was supplied by Hubei Chemical Fiber Co. Ltd. (Xiangyang, China). Its viscosity-average molecular weight (Mv) determined in LiOH/urea aqueous solution by viscometry and calculated from the equation [η] = 3.72 × 10−2Mη0.77 was 1.0 × 105 g/mol (Cai & Zhang, 2006). Other analytical grade chemicals used were obtained from Sinopharm Chemical Reagent Co., Ltd.

CrI002 =

I002 − Iam × 100% I002

(1)

where I002 and Iam refer to the maximum intensity of 002 peak at 2θ = 22.7° and the minimum between the 002 and 101 peaks (2θ = 16.0°) as the amorphous peak, respectively. Scanning electron microscopy (SEM) images were performed on a Hitachi S-570 (Tokyo, Japan) microscope at an accelerating voltage of 15 kV. The samples were immersed in distilled water to reach an equilibrium swelling state. Then the wet samples were frozen in liquid nitrogen, snapped immediately and finally freeze-dried. The fracture section was sputtered with gold for observation. Optical transmittance (Tr) of the films was recorded on a UV-1601 (Japan) UV–vis spectrometer with the wavelength ranging from 200 to 800 nm (Li, Zhou, & Zhang, 2009). The thermal stability was determined by thermal analyzer (6300TG-DSC, PE, USA) from 30 °C to 600 °C with a heating rate of 10 °C/min under nitrogen atmosphere. The mechanical properties were determined on a universal testing machine (CMT6503, Shenzhen SANS TEST machine Co., Ltd., China) at a speed of 1 mm/min−1. Here, at least five parallels were determined for each sample and the average value was reported.

2.2. Preparation of CNW CNW were extracted from the cotton linter pulp using a modified version of the acid hydrolysis procedure previously reported (Bondeson, Mathew, & Oksman, 2006). Briefly, 10 g cellulose were mixed with 150 mL of 63% sulfuric acid solution and hydrolyzed at 50 °C for 2 h under vigorous stirring to obtain CNW. The resultant suspension was diluted over 10-folds and centrifuged at 21288g by using Avanti J-25 (JA-18, Beckman Coulter, America) to remove excessive acid until the supernatant become turbid. Then, the suspension was dialyzed against pure water until the pH value constant. The samples were sonicated under an ultrasonic cell disruptor (JY98-3D, Ningbo Scientz Biotechnology Co., Ltd., China) at 600 w for 30 min in an ice bath. Finally, the CNW suspension was centrifuged at 5322g to discard the precipitate and stored at 4 °C before further use. The concentration of CNW suspension calculated by the weight ratio of solid and suspension after freeze-drying was 1.7%.

2.5. Swelling property A gravimetric method (Gabrielii, Gatenholm, Glasser, Jain, & Kenne, 2000; Khan et al., 2012) was used to study the swelling property of the nanocomposite films in pure water or 0.9 wt% NaCl solution at 25 °C. Small pieces of films were placed in glass dishes with known weight. 20 mL of pure water (or 0.9 wt% NaCl solution) was added, left for 30 min and then carefully removed, leaving the sample on the dish. The dish loaded samples was wiped dry with filter paper carefully and weighed. Each test was repeated for three times. The swelling percentage (S) was calculated as follows:

2.3. Preparation of chitosan-xylan/CNW nanocomposite films According to previous researches, chitosan–xylan mixtures with ratios of 1:1, 1:2, 1:3 and 1:4 were prepared (Li, Shi, Wang, & Du, 2011; Wu, Hu, Wei, Du, Shi, Deng et al., 2014). However, xylans are excessive when the ratios are 1:3 and 1:4. Chitosan–xylan mixture with ratio of 1:2 was selected for the preparation of chitosan-xylan/CNW nanocomposite films as a relatively high ratio would be more helpful for the interaction between chitosan and xylan. 4 g chitosan powder was added into 100 mL 2% (v/v) glacial acetic acid and stirred for 4 h to obtain transparent solution. Then 8 g xylan was added and stirred to obtain a homogeneous solution. Calculated amounts of CNW were added to the above chitosan-xylan solution and stirred for 2 h. The mixture of chitosan-xylan/CNW was sonicated for 20 min, and then centrifuged at 5322g for 10 min to remove the bubbles. The obtained solutions were poured into a small plastic tray and were heated in an oven at 90 °C for 1 h, followed by heated at 40 °C for 8 h, the film formed as the solvent

S=

Ws − Wd × 100 Wd

(2)

Where Wd and Ws were the weight of the dry films and the water swollen films, respectively. 2.6. Antibacterial activity The antibacterial activity of chitosan, xylan, CX and CX/CNW12 against Gram-negative E.coli and Gram-positive S.aureus was evaluated using a modified version of the optical method (Dominguez, de la Rosa & Borobio, 2001; Shankar, Reddy, Rhim, & Kim, 2015). 0.1 g of the above samples were sterilized under an ultraviolet radiation lamp for 30 min, and incubated with 20 mL of 106 cfu/mL E.coli or S.aureus in 67

Carbohydrate Polymers 184 (2018) 66–73

Y. Bao et al.

were calculated to be approximately 68.1% and 90.9% using Eq. (1). The higher crystallinity of CNW is anticipated to improve the properties of the nanocomposite films. The TGA curves of native cellulose and CNW are shown in Fig. S2. A weight loss about 5% under 100 °C could be attributed to the moisture release. A rapid decrease from 210 °C to 380 °C resulted from the thermal decomposition of cellulose backbone (Zhou, Long, Meng, Chen, Li, & Zhang, 2015). T50 (decomposition temperature at 50% weight loss) of native cellulose and CNW were 350 °C and 334 °C, respectively. The results revealed that CNW possessed lower thermal stability than that of native cellulose, and this was owing to the sulfated groups in CNW (Roman & Winter, 2004). The final residues of CNW were higher than native cellulose may ascribe to the dehydration effect of the sulfate group in CNW (Jiang & Hsieh, 2013; Wang, Ding, & Cheng, 2007). These results evidenced the successful preparation of CNW.

meat-peptone broth at 37 °C for 12–24 h under mild shaking. The inhibitory effect was assessed by measuring the optical density of the cultured medium at 600 nm of E.coli and 450 nm of S.aureus periodically. The experiment was carried out in triplicate. 2.7. Antioxidant activity Antioxidant activity of chitosan, xylan, CX and CX/CNW12 was determined by ABTS method using Duygu Altiok’s (Altiok, Altiok, & Tihminlioglu, 2010). The ABTS+ radical was generated by a mixture of 7 mM ABTS and 2.45 mM K2S8O2 placed under the dark for 12 h. The ABTS+ solution was diluted with phosphate buffered saline (PBS, pH = 7.4) to adjust the absorbance to 0.70 ± 0.05 at 734 nm 10 mg of xylan, chitosan, CX and CX/CNW12 samples were added to 5 mL of diluted ABTS+ solution, respectively. The solution with samples was shaken at 30 °C shielded from the light for 30 min, and a solution without samples under the same conditions was recorded as control. The absorbance was determined at 734 nm. The ABTS+ radicalscavenging activity could be expressed as follows:

A − As Scanvenging activity(%) = 0 × 100 A0

3.2. Structure and morphology of the nanocomposite films Fig. 2A displays the FT-IR spectra of chitosan, xylan, CNW, CX and nanocomposite films. For chitosan (a), the peaks at 1659 cm−1 and 1597 cm−1 are owing to the amide I and primary amino group, respectively (Li, Shi, Wang, & Du, 2011). For xylan, the broad band at 1043 cm−1 representing the stretching and bending vibration of CeO, CeC or CeOH (Wu, Hu, Wei, Du, Shi et al., 2014). In the spectrum of CX (c), the characteristic peaks of chitosan at 1659 cm−1 and 1597 cm−1 disappeared, and new bands at 1560 cm−1 and 1716 cm−1 were observed, it could be explained by the interaction of chitosan amino groups with the reducing end of xylan to form Schiff base (C]N double bond) (Sedlmeyer, 2011). In the spectra of nanocomposite films (d–g), the characteristic peaks of CNW (saccharide and CeH stretching bands at 1161 cm−1and 1063 cm−1) and CX (C]N bands at 1560 cm−1 and 1716 cm−1) were observed. The band of nanocomposite films at 3300–3550 cm−1 shifted in comparison with CNW and CX, which could be due to the electrostatic interactions between the anionic sulfate groups of CNW and the amine groups of chitosan, or hydrogen bonds between the OH groups of CNW and the ammonium groups of chitosan (de Mesquita, Donnici, & Pereira, 2010; Karim, Mathew, Grahn, Mouzon, & Oksman, 2014; Li, Zhou & Zhang, 2009). These interactions play an important role in reinforcing the properties of the nanocomposite films. Fig. 2B displays the XRD patterns of chitosan, xylan, CNW, CX and nanocomposite films. The characteristic peaks of chitosan and xylan were observed from Fig. 2Ba and b at 2θ = 10.2°, 19.9°, 29.4° and 18.7°, respectively (Trimukhe & Varma, 2008; Wu, Du, Hu, Shi, & Zhang, 2013). However, only one new peak in the XRD pattern of CX at 19.1° was observed with the crystallinity sharply decreased, indicating that new crystalline substance was formed. In the XRD patterns of the nanocomposite films, the broad peak at 19.1° signified an amorphous structure of CX, and the sharp peak at 2θ = 22.7° signified a hydrated crystalline structure of CNW (Haafiz, Hassan, Zakaria, Inuwa, & Islam, 2013; Wu, Hu, Wei, Du, Shi et al., 2014). And the intensity was increased with an increment of CNW contents. Meanwhile, the typical broad peak of amorphous CX at 2θ = 19.1° overlaid with the sharp peak

(3) +

Where As was the absorbance of sample with ABTS solution, A0 was the absorbance of the control ABTS+ solution. The experiments were carried out in triplicate. 2.8. Statistical analysis Statistics of tensile strength, elongation at break, swelling percentage and antioxidant activity were represented as the mean batches ± SD and analyzed using SPSS statistical software by analysis of variance (ANOVA). Statistical significance was determined by Duncan’s multiple range test (P < 0.05), and values not sharing a common superscript (a, b, c, d and e) differ significantly (Zhang et al., 2017). 3. Results and discussion 3.1. Characterization of CNW Fig. 1b shows the flow birefringence of aqueous CNW viewed through two cross-polarizers. When the concentration of CNW reached a critical value, an anisotropic phase appeared under the polarized optical microscope (POM) (Fig. 1c). The phenomenon indicated a chiral nematic liquid crystalline alignment and revealed the existence of CNW (Bondeson, Mathew, & Oksman, 2006). TEM image (Fig. 1d) also confirmed the successful preparation of CNW, as the whiskers displayed rod-like morphology with length of 200–450 nm and width of ∼20 nm (the aspect ratio was larger than 10). XRD patterns of native cellulose and CNW are displayed in Fig. S1, both of them showed a strong peak at 2θ = 22.7° and two overlapped weaker peaks at 2θ = 14.8° and 16.4°, corresponding to the typical XRD pattern of cellulose I (Sun, Chen, Jiang, & Lynch, 2015). The crystallinity of native cellulose and CNW

Fig. 1. Optical photograph of CNW suspension in water (a), flow birefringence of CNW in water observed between two crossed polarizers (b), POM photograph of CNW suspension in water (c), TEM image of CNW dispersed in water (d).

68

Carbohydrate Polymers 184 (2018) 66–73

Y. Bao et al.

Fig. 2. FT-IR spectra (A) and XRD patterns (B) of chitosan (a), xylan (b), CX (c), CX/CNW4 (d), CX/ CNW8 (e), CX/CNW12 (f), CX/CNW16 (g), and CNW (h).

nanocomposite films in the wavelength of 200–800 nm determined by UV–vis spectrometer, and Tr values at 800 nm is presented in Fig. 4b. It’s obvious that the Tr of the nanocomposite films decreased with the increment of CNW. Absorption of ultra-violet light at the wavelength from 200 to 500 nm was due to the brown color of the nanocomposite films. Tr values at 800 nm decreased slightly from 88% to 67% with CNW contents increased from 0 to 16 wt%. The nanocomposite films exhibited a Tr of 76% with 12 wt% contents of CNW, the relatively high transmittance indicated good miscibility between CNW and the matrix in consequence of their strong interaction (Huang, Zhang, Yang, Zhang, & Xu, 2013).The Tr value of nanocomposite films with 16 wt% CNW addition significantly decreased to 67%, due to the slight microphase separation cause by the aggregation of excessive CNW within the nanocomposite films (Li, Zhou, & Zhang, 2009). The TGA analyses were performed to study the thermal properties of the nanocomposite films. TGA curves of chitosan and xylan are shown in Fig. S3, and both of them displayed two stage thermal decomposition processes, the first stage weight loss under 110 °C is related to the moisture release from the samples, and the second stage corresponds to the decomposition of chitosan (range from 250 to 350 °C) or xylan

of CNW at 22.7°. These results indicated that CNW reinforced CX films showed a combination of amorphous and crystalline peaks, and the increased crystallinity may lead to enhanced mechanical properties. SEM cross-section images of CX and the nanocomposite films were showed in Fig. 3. It was obvious that all of the films displayed a microporous structure, and in the nanocomposite films, fibrous materials were observed and uniform dispersed in the matrices, evidenced the existence of CNW. Besides, the pore structure became relatively denser with the contents of CNW increased to 12% in the nanocomposite films. The difference of pore structure may be ascribed to the hydrogen bonds and electrostatic interactions between CNW and chitosan, tightening the network and resulting in smaller pores (Chen, Liu, & Zeng, 2016; Karim, Mathew, Grahn, Mouzon, & Oksman, 2014). However, the pore structure was damaged with fractures appearing when the contents of CNW further improved to 16%, and it may be caused by the aggregation of excessive amounts of CNW within the nanocomposite films. 3.3. Optical, thermal and mechanical properties of the nanocomposite films Fig. 4a shows the optical transparency (Tr) values of CX and the

Fig. 3. SEM cross-section images of CX (a), CX/ CNW4 (b), CX/CNW12 (c), and CX/CNW16 (d).

69

Carbohydrate Polymers 184 (2018) 66–73

Y. Bao et al.

Fig. 4. Optical transmittance (Tr) of CX, CX/CNW4, CX/CNW8, CX/CNW12 and CX/CNW16 from 200 to 800 nm (a) and dependence of Tr on CNW content at 800 nm (b).

Fig. 5. TGA curves (a) and Stress (σ)/strain (ε) curves (b) of CX, CX/CNW4, CX/CNW8, CX/CNW12 and CX/CNW16.

decrease in tensile strength and elongation at break for CX/CNW16 may be resulted from the aggregation of excessive CNW and the stiffness of CNW (Li, Zhou, & Zhang, 2009).

(range from 200 to 350 °C) backbone (Corazzari et al., 2015; Sousa, Ramos, Evtuguin, & Gamelas, 2016). A similar thermal behavior at the temperature range from 250 to 350 °C was found in TGA curves of CX and nanocomposite films (Fig. 5a). T50 of CX, CX/CNW4, CX/CNW8, CX/CNW12 and CX/CNW16 were at 337 °C, 333 °C, 332 °C, 331 °C and 336 °C, respectively. Moreover, it was observed that no obvious differences were observed in the TGA curves of CX and CX/CNWs, and different from the curve of CNW (Fig. S2). Meanwhile, the temperature at maximum decomposition rate of CX, CX/CNW4, CX/CNW8, CX/ CNW12 and CX/CNW16 displayed in the DTG curves (Fig. S4) were hardly changed and observed at 293 °C, 293 °C, 290 °C, 293 °C, 288 °C, respectively. These results proved a union dispersion of CNW in CX matrix, demonstrating that the addition of CNW did not affect the thermal stability of the CX matrices. Tensile tests were used to study the reinforcing effect of the CNW on the mechanical properties of CX matrix. The tensile stress-strain curves, tensile strength (σ) and elongation at break (ε) of the films are presented in Figs. Figure 5b and S5. Tensile strength of the films increased from 4.90 ± 0.36 to 16.04 ± 0.22 MPa with increments of the content of CNW from 0 to 12 wt%. However, the tensile strength decreased to 12.76 ± 0.24 MPa when further improved the contents of CNW to 16%. The increase corresponds to a gain in tensile strength of 27, 184, 227 and 160% compared to CX, respectively. In addition, the elongation at break presented a similar tendency as the tensile strength, increased from 6.47 ± 0.22% to 11.49 ± 0.21% and then decreased to 6.56 ± 0.40% with the same contents of CNW. These results manifested that the introduction of CNW had an effect on reinforcing the mechanical properties of the nanocomposite films, and this may be related to the effective stress transfer at the CNW-chitosan polymer interface (Rubentheren, Ward, Chee, & Nair, 2015). The negatively charged sulfate groups of CNW and the positive charged amine groups of chitosan could form an interface between the polymer matrix and the CNW nanofiller (de Mesquita, Donnici, & Pereira, 2010). However, the

3.4. Swelling property The swelling property of CX and the nanocomposite films in pure water and 0.9 wt% NaCl solutions is shown in Fig. 6A and B, respectively. The S of CX and nanocomposite films with 4, 8, 12 and 16 wt% contents of CNW in pure water was 81.5 ± 0.36%, 67.2 ± 0.62%, 64.7 ± 0.6%, 60.4 ± 0.70% and 52.6 ± 0.55%, respectively, displaying a decrease tendency with the increment of CNW. Meanwhile, the S in 0.9 wt% NaCl solutions presented the same tendency from 69.6 ± 0.60% to 41.2 ± 0.95%. The results was related to the lower hydrophilicity of CNW and the interaction between CNW and the matrix, the introduction of CNW will provide an interpenetrated network within the matrix, preventing the water swelling property of the CX films (Khan et al., 2012; Li, Zhou, & Zhang, 2009). Moreover, nanocomposite films could absorb much more pure water than NaCl solution, and it could be explained by the decrease of osmotic pressure between the internal polymeric network and the external solution (Wu, Hu, Wei, Du, Shi, Deng et al., 2014). CX showed higher swelling percentage than that of nanocomposite films in both conditions, proved an increased stability for nanocomposite films in aqueous solution. 3.5. Antibacterial activity The antibacterial activity of chitosan, xylan, CX and CX/CNW12 against Gram-negative E.coli and Gram-positive S.aureus was evaluated by determining the optical density (OD, at 600 nm and 450 nm for E.coli and S.aureus, respectively) of culture medium including the test nanocomposite films and microorganisms. The microorganism growth will increase the OD values; therefore, lower absorbance represents better 70

Carbohydrate Polymers 184 (2018) 66–73

Y. Bao et al.

Fig. 6. Swelling percentage of CX (0% content of CNW) and the nanocomposite films with different CNW contents (4%, 8%, 12%, and 16%) in pure water (A) and 0.9% NaCl solution (B).

Fig. 7. Antibactarial activity of the control, chitosan, xylan, CX and CX/CNW12 against E. coli (a) S. aureus (b).

antibacterial activity of the samples. As demonstrated in Fig. 7, the OD values of both the xylan samples and the control increased continuously during the incubation, indicating that xylan did not show any antibacterial activity against E.coli and S.aureus. However, the OD values of chitosan, CX and CX/CNW12 samples were greatly suppressed, and they were almost the same for CX and CX/CNW12 samples. As previous researches have convinced that the antibacterial activity of chitosan was attributed to the interaction between the polycationic amino groups and the anionic substance on the surface of bacterial cells that will change the cell membrane permeability (Krajewska, Wydro, & Jańczyk, 2011), and the formed film over the cell membrane surface that will prevent the nutrients from entering into the cell (Liu, Du, Wang, & Sun, 2004). Besides, CX possesses the ability of destroying the membrane of bacterial cells to inhibit their growth due to excellent surfactant properties (Li, Shi, Wang, & Du, 2011). Therefore, the antibacterial activity mainly originated from the chitosan and the addition of CNW into the nanocomposite films will not affect it.

Fig. 8. ABTS+ scavenging activity (%) of chitosan, xylan, CX and CX/CNW12.

4. Conclusion CX/CNW nanocomposite films with antibacterial and antioxidant activity were successfully prepared by the introduction of CNW into the chitosan-xylan matrix. It was found that by adding a mass fraction of CNW to 12 wt%, the tensile strength and elongation at break of the nanocomposite films were increased significantly. The swelling percentage of the nanocomposite films decreased with the increment of CNW contents, proved an increased stability for nanocomposite films in aqueous solution. In addition, the nanocomposite films possessed good antibacterial activity against S. aureus and E. coli and antioxidant activity. The prepared chitosan-xylan/CNW nanocomposite films, combined the antibacterial property of chitosan, the antioxidant property of xylan, and good mechanical property of CNW, could be potentially applied in food and health-related areas.

3.6. Antioxidant activity The antioxidant activity of chitosan, xylan, CX and CNW12 was investigated by ABTS method. As shown in Fig. 8, Chitosan and xylan displayed an ABTS+ scavenging activity of 10.7 ± 1.57% and 94.1 ± 0.79%, respectively. The high ABTS+ scavenging activity of xylan might be the reason that xylan isolated from the corn cobs was a water-soluble reductive polysaccharide branched with arabinose and uronic acid (Li, Shi, Wang, & Du, 2011). Previous research has reported that chitosan-xylan conjugates prepared by heating would possess relatively good antioxidant activity owing to the sugar-amine condensation of chitosan and xylan, and the scavenging activity of CX was 48.5 ± 0.97% (Li, Shi, Jin, Ding, & Du, 2013). It was also found that the nanocomposite film with 12 wt% contents of CNW has a scavenging activity of 41.5 ± 1.57%, suggested that the introduction of CNW has little influence on the antioxidant activity of the nanocomposite films. 71

Carbohydrate Polymers 184 (2018) 66–73

Y. Bao et al.

Acknowledgments

Kenny, O., Smyth, T. J., Walsh, D., Kelleher, C. T., Hewage, C. M., & Brunton, N. P. (2014). Investigating the potential of under-utilised plants from the Asteraceae family as a source of natural antimicrobial and antioxidant extracts. Food Chemistry, 161(11), 79–86. Khan, A., Khan, R. A., Salmieri, S., Le, T. C., Riedl, B., Bouchard, J., et al. (2012). Mechanical and barrier properties of nanocrystalline cellulose reinforced chitosan based nanocomposite films. Carbohydrate Polymers, 90(4), 1601–1608. Krajewska, B., Wydro, P., & Jańczyk, A. (2011). Probing the modes of antibacterial activity of chitosan. Effects of pH and molecular weight on chitosan interactions with membrane lipids in Langmuir films. Biomacromolecules, 12(11), 4144–4152. Li, Q., Zhou, J., & Zhang, L. (2009). Structure and properties of the nanocomposite films of chitosan reinforced with cellulose whiskers. Journal of Polymer Science Part B Polymer Physics, 47(11), 1069–1077. Li, X., Shi, X., Wang, M., & Du, Y. (2011). Xylan chitosan conjugate-a potential food preservative. Food Chemistry, 126(2), 520–525. Li, X., Shi, X., Jin, Y., Ding, F., & Du, Y. (2013). Controllable antioxidative xylan–chitosan Maillard reaction products used for lipid food storage. Carbohydrate Polymers, 91(1), 428–433. Liu, H., Du, Y., Wang, X., & Sun, L. (2004). Chitosan kills bacteria through cell membrane damage. International Journal of Food Microbiology, 95(2), 147–155. Liu, D., Dong, Y., Bhattacharyya, D., & Sui, G. (2017). Novel sandwiched structures in starch/cellulose nanowhiskers (CNWs) composite films. Composites Communications, 4, 5–9. Martínez-Sanz, M., Lopez-Rubio, A., & Lagaron, J. M. (2013). High-barrier coated bacterial cellulose nanowhiskers films with reduced moisture sensitivity. Carbohydrate Polymers, 98(1), 1072–1082. Martínez-Sanz, M., Vicente, A. A., Gontard, N., Lopez-Rubio, A., & Lagaron, J. M. (2015). On the extraction of cellulose nanowhiskers from food by-products and their comparative reinforcing effect on a polyhydroxybutyrate-co-valerate polymer. Cellulose, 22(1), 535–551. Mikkonen, K. S., & Tenkanen, M. (2012). Sustainable food-packaging materials based on future biorefinery products: Xylans and mannans. Trends in Food Science & Technology, 28(2), 90–102. Ngah, W. S. W., Teong, L. C., & Hanafiah, M. A. K. M. (2011). Adsorption of dyes and heavy metal ions by chitosan composites: A review. Carbohydrate Polymers, 83(4), 1446–1456. Njuguna, J., Pielichowski, K., & Desai, S. (2010). Nanofiller-reinforced polymer nanocomposites. Polymers for Advanced Technologies, 19(8), 947–959. Pal, K., Behera, B., Roy, S., Ray, S. S., & Thakur, G. (2013). Chitosan based delivery systems on a length scale: Nano to macro. Soft Materials, 11(2), 125–142. Prabaharan, M. (2015). Chitosan-based nanoparticles for tumor-targeted drug delivery. International Journal of Biological Macromolecules, 72(5), 1313–1322. Qi, H., Cai, J., Zhang, L., & Kuga, S. (2009). Properties of films composed of cellulose nanowhiskers and a cellulose matrix regenerated from alkali/urea solution. Biomacromolecules, 10(6), 1597–1602. Roman, M., & Winter, W. T. (2004). Effect of sulfate groups from sulfuric acid hydrolysis on the thermal degradation behavior of bacterial cellulose. Biomacromolecules, 5(5), 1671–1677. Rubentheren, V., Ward, T. A., Chee, C. Y., & Nair, P. (2015). Physical and chemical reinforcement of chitosan film using nanocrystalline cellulose and tannic acid. Cellulose, 22(4), 2529–2541. Sedlmeyer, F. B. (2011). Xylan as by-product of biorefineries: Characteristics and potential use for food applications. Food Hydrocolloids, 25(8), 1891–1898. Shankar, S., Reddy, J. P., Rhim, J. W., & Kim, H. Y. (2015). Preparation, characterization, and antimicrobial activity of chitin nanofibrils reinforced carrageenan nanocomposite films. Carbohydrate Polymers, 117, 468–475. Silva, T. C. F., Habibi, Y., Colodette, J. L., & Lucia, L. A. (2011). The influence of the chemical and structural features of xylan on the physical properties of its derived hydrogels. Soft Matter, 7(3), 1090–1099. Sousa, S., Ramos, A., Evtuguin, D. V., & Gamelas, J. A. F. (2016). Xylan and xylan derivatives—Their performance in bio-based films and effect of glycerol addition. Industrial Crops & Products, 94, 682–689. Sun, L., Chen, J. Y., Jiang, W., & Lynch, V. (2015). Crystalline characteristics of cellulose fiber and film regenerated from ionic liquid solution. Carbohydrate Polymers, 118, 150–155. Sunkyu, P., Baker, J. O., Himmel, M. E., Parilla, P. A., & Johnson, D. K. (2010). Cellulose crystallinity index: Measurement techniques and their impact on interpreting cellulase performance. Biotechnology for Biofuels, 3, 1–10. Trimukhe, K. D., & Varma, A. J. (2008). A morphological study of heavy metal complexes of chitosan and crosslinked chitosans by SEM and WAXRD. Carbohydrate Polymers, 71(4), 698–702. Wang, N., Ding, E., & Cheng, R. (2007). Thermal degradation behaviors of spherical cellulose nanocrystals with sulfate groups. Polymer, 48(12), 3486–3493. Wu, S., Du, Y., Hu, Y., Shi, X., & Zhang, L. (2013). Antioxidant and antimicrobial activity of xylan–chitooligomer–zinc complex. Food Chemistry, 138(2), 1312–1319. Wu, S., Hu, J., Wei, L., Du, Y., Shi, X., & Zhang, L. (2014). Antioxidant and antimicrobial activity of Maillard reaction products from xylan with chitosan/chitooligomer/glucosamine hydrochloride/taurine model systems. Food Chemistry, 148, 196–203. Wu, S., Hu, J., Wei, L., Du, Y., Shi, X., Deng, H., et al. (2014). Construction of porous chitosan–xylan–TiO2 hybrid with highly efficient sorption capability on heavy metals. Journal of Environmental Chemical Engineering, 2(3), 1568–1577. Ying, G., Jing, B., Feng, P., Zhang, X. M., & Sun, R. C. (2014). High strength of hemicelluloses based hydrogels by freeze/thaw technique. Carbohydrate Polymers, 101(1), 272–280. Zeng, D., Wu, J., & Kennedy, J. F. (2008). Application of a chitosan flocculant to water treatment. Carbohydrate Polymers, 71(1), 135–139.

This work was supported by the National Key Research and Development Program of China (2017YFD0400200), the National Natural Science Foundation of China (31701560, 31671820), the Basic Applied Research Project of Wuhan City (2016020101010095), Agricultural Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2013-OCRI). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.carbpol.2017.12.051. References Aider, M. (2010). Chitosan application for active bio-based films production and potential in the food industry: Review. LWT – Food Science and Technology, 43(6), 837–842. Altiok, D., Altiok, E., & Tihminlioglu, F. (2010). Physical, antibacterial and antioxidant properties of chitosan films incorporated with thyme oil for potential wound healing applications. Journal of Materials Science-Materials in Medicine, 21(7), 2227–2236. Ayoub, A., Venditti, R. A., Pawlak, J. J., Salam, A., & Hubbe, M. A. (2013). Novel hemicellulose–chitosan biosorbent for water desalination and heavy metal removal. ACS Sustainable Chemistry & Engineering, 1(9), 1102–1109. Azizi Samir, M. A., Alloin, F., & Dufresne, A. (2005). Review of recent research into cellulosic whiskers, their properties and their application in nanocomposite field. Biomacromolecules, 6(2), 612–626. Bondeson, D., Mathew, A., & Oksman, K. (2006). Optimization of the isolation of nanocrystals from microcrystalline celluloseby acid hydrolysis. Cellulose, 13(2), 171–180. Cai, J., & Zhang, L. (2006). Unique gelation behavior of cellulose in NaOH/Urea aqueous solution. Biomacromolecules, 7(1), 183–189. Carocho, M., & Ferreira, I. C. (2013). A review on antioxidants, prooxidants and related controversy: Natural and synthetic compounds, screening and analysis methodologies and future perspectives. Food & Chemical Toxicology, 51(1), 15–25. Chen, Y., Liu, W. Y., & Zeng, G. S. (2016). Stimulus-responsive hydrogels reinforced by cellulose nanowhisker for controlled drug release. RSC Advances, 6, 87422–87432. Corazzari, I., Nisticò, R., Turci, F., Faga, M. G., Franzoso, F., Tabasso, S., et al. (2015). Advanced physico-chemical characterization of chitosan by means of TGA coupled on-line with FTIR and GCMS: Thermal degradation and water adsorption capacity. Polymer Degradation & Stability, 112, 1–9. Devlieghere, F., Vermeulen, A., & Debevere, J. (2004). Chitosan: Antimicrobial activity, interactions with food components and applicability as a coating on fruit and vegetables. Food Microbiology, 21(6), 703–714. Dominguez, M. C., de la Rosa, M., & Borobio, M. V. (2001). Application of a spectrophotometric method for the determination of post-antibiotic effect and comparison with viable counts in agar. Journal of Antimicrobial Chemotherapy, 47(4), 391–398. Dotto, G. L., Pinto, L. A., Hachicha, M. A., & Knani, S. (2015). New physicochemical interpretations for the adsorption of food dyes on chitosan films using statistical physics treatment. Food Chemistry, 171(171), 1–7. Duan, J., Liang, X., Cao, Y., Wang, S., & Zhang, L. (2015). High strength chitosan hydrogels with biocompatibility via new avenue based on constructing nanofibrous architecture. Macromolecules, 48(8), 2706–2714. Ebringerova, A., & Heinze, T. (2000). Xylan and xylan derivatives – biopolymers with valuable properties, 1 – naturally occurring xylans structures, procedures and properties. Macromolecular Rapid Communications, 21(9), 542–556. Eichhorn, S. J. (2011). Cellulose nanowhiskers: Promising materials for advanced applications. Soft Matter, 7(2), 303–315. Gümüskaya, E., Usta, M., & Kirci, H. (2003). The effects of various pulping conditions on crystalline structure of cellulose in cotton linters. Polymer Degradation & Stability, 81(3), 559–564. Gabrielii, I., Gatenholm, P., Glasser, W. G., Jain, R. K., & Kenne, L. (2000). Separation, characterization and hydrogel-formation of hemicellulose from aspen wood. Carbohydrate Polymers, 43(4), 367–374. Haafiz, M. K. M., Hassan, A., Zakaria, Z., Inuwa, I. M., & Islam, M. S. (2013). Physicochemical characterization of cellulose nanowhiskers extracted from oil palm biomass microcrystalline cellulose. Materials Letters, 113(12), 87–89. Hajji, S., Salem, S. B., Hamdi, M., Jellouli, K., Ayadi, W., Nasri, M., et al. (2017). Nanocomposite films based on chitosan–poly(vinyl alcohol) and silver nanoparticles with high antibacterial and antioxidant activities. Process Safety & Environmental Protection, 111, 112–121. Herrera, N. V., Mathew, A. P., Wang, L. Y., & Oksman, K. (2011). Randomly oriented and aligned cellulose fibres reinforced with cellulose nanowhiskers, prepared by electrospinning. Plastics Rubber and Composites, 40(2), 57–64. Huang, Y., Zhang, L., Yang, J., Zhang, X., & Xu, M. (2013). Structure and properties of cellulose films reinforced by chitin whiskers. Macromolecular Materials and Engineering, 298(3), 303–310. Jiang, F., & Hsieh, Y. L. (2013). Chemically and mechanically isolated nanocellulose and their self-assembled structures. Carbohydrate Polymers, 95(1), 32–40. Karim, Z., Mathew, A. P., Grahn, M., Mouzon, J., & Oksman, K. (2014). Nanoporous membranes with cellulose nanocrystals as functional entity in chitosan: Removal of dyes from water. Carbohydrate Polymers, 112(2), 668–676.

72

Carbohydrate Polymers 184 (2018) 66–73

Y. Bao et al.

kinetics analysis of pyrolysis of hemicellulose, cellulose, and lignin in TGA and macro-TGA. RSC Advances, 5(34), 26509–26516. de Mesquita, J. P., Donnici, C. L., & Pereira, F. V. (2010). Biobased nanocomposites from layer-by-layer assembly of cellulose nanowhiskers with chitosan. Biomacromolecules, 11(2), 473–480.

Zhang, H., Yang, M., Luan, Q., Tang, H., Huang, F., Xiang, X., et al. (2017). Cellulose anionic hydrogels based on cellulose nanofibers as natural stimulants for seed germination and seedling growth. Journal of Agricultural & Food Chemistry, 65(19), 3785–3791. Zhou, H., Long, Y., Meng, A., Chen, S., Li, Q., & Zhang, Y. (2015). A novel method for

73