Effects of chitin nano-whiskers on the antibacterial and physicochemical properties of maize starch films

Accepted Manuscript Title: Effects of chitin nano-whiskers on the antibacterial and physicochemical properties of maize starch films Author: Yang Qin. Shuangling Zhang Jing Yu Jie Yang Liu Xiong Qingjie Sun PII: DOI: Reference:

S0144-8617(16)30348-4 http://dx.doi.org/doi:10.1016/j.carbpol.2016.03.095 CARP 10932

To appear in: Received date: Revised date: Accepted date:

30-11-2015 28-3-2016 29-3-2016

Please cite this article as: Zhang, Yang Qin Shuangling., Yu, Jing., Yang, Jie., Xiong, Liu., & Sun, Qingjie., Effects of chitin nano-whiskers on the antibacterial and physicochemical properties of maize starch films.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.03.095 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

Effects of chitin nano-whiskers on the antibacterial and physicochemical properties of maize starch films

Yang Qin. Shuangling Zhang, Jing Yu, Jie Yang, Liu Xiong, Qingjie Sun*

School of Food Science and Engineering, Qingdao Agricultural University (Qingdao, Shandong Province, 266109, China) *Correspondence

author

(Tel:

86-532-88030448,

Fax:

86-532-88030449,

e-mail:

[email protected]), School of Food Science and Engineering, Qingdao Agricultural University, 266109, 700 Changcheng Road, Chengyang District, Qingdao, China.

Highlight 

Effects of chitin nano-whiskers (CNWs) on maize starch–based films were investigated.



Mechanical, opacity and barrier properties of the films were increased significantly.



The To, Tp and ΔH of the nanocomposite films increased with CNW content increased.



Films exhibit stronger antimicrobial activity against L. monocytogenes than E. coli.

Abstract We investigated the effects of chitin nano-whiskers (CNWs) on the antibacterial and physiochemical properties of maize starch–based films. The microstructures, crystalline structures, and thermal, mechanical and barrier properties of the nanocomposite films were characterized by using transmission electron microscopy, X-ray diffraction analysis, thermogravimetric, differential scanning calorimeter, and texture profile analysis. The tensile strength of the maize starch films increased from 1.64 MPa to 3.69 MPa (P < 0.05) after CNW reinforcement with up to 1%. The water vapor permeability of the nanocomposite films decreased from 5.32 × 10–12 to 2.22 × 10–12 g m–1s–1Pa–1 with the CNW content increasing from 0% to 2%. The onset temperature, peak 1

temperature and the gelatinization enthalpy of the films containing CNWs were higher than those of the pure starch films. Furthermore, the nanocomposite films exhibited strong antimicrobial activity against Gram-positive L. monocytogenes but not against Gram-negative Escherichia coli. Keywords: Maize starch film, Chitin nano-whiskers, Antibacterial activity, Mechanical properties 1. Introduction Concern about the environment and the depletion of natural resources caused by petroleum-based plastics has focused attention to the development of environmentally benign polymer nanocomposites for applications in the food, cosmetics, and pharmaceutical industries. Renewable and abundantly available biopolymers are the most viable alternative for producing green materials in the near future. Nature has provided various natural biopolymers, such as polysaccharides (starch, cellulose, and chitosan) and proteins (soy protein, wheat protein, casein, and gelatin). Utilization of natural biopolymers for making biodegradable packaging films, edible film, and coating materials has increased considerably in the literature (Rhim, Park, & Ha, 2013). Among such natural biopolymers, starch is one of the most commonly used raw materials to prepare biodegradable films or edible packaging films, because it is an inexpensive renewable source that is widely available and relatively easy to handle. Unfortunately, poor performances such as lower water vapor barrier, relatively lower mechanical properties, and processing difficulty are the main limitations of these biopolymer-based films. Thus, to overcome above problems, a number of studies have been performed by reinforcing nanofiller materials (Martins et al., 2013; Rhim et al., 2013). Inorganic (metallic nanoparticles and nano-clays) and organic synthetic material (carbon nanotubes, nanographite) nanofillers have been used as reinforcing materials (Chandrasekaran, Seidel, & Schulte, 2013; Du et al., 2014). Polysaccharides such as starch, cellulose, and chitin are potential renewable sources of nano-size reinforcement because of their completely biodegradable nature. Promising fillers are biopolymer nanocrystals, in which the nano-size fillers can impart enhanced mechanical and barrier properties, such as tensile strength, flexibility, and the modulus of elasticity. Following this strategy, nanocomposite materials have been prepared from plasticized starch reinforced with starch nanoparticles (Angellier, Molina-Boisseau, Dole, & Dufresne, 2006; Dai, Qiu, Xiong, & Sun, 2015; Shi, Wang, Li, & Adhikari, 2013), cellulose nanocrystals (Chang, Jian, Yu, & Ma, 2

2010a; Reddy & Rhim, 2014; Savadekar & Mhaske, 2012), and protein reinforced with chitosan nanoparticles (Hosseini, Rezaei, Zandi, & Farahmandghavi, 2015). Moreover, adding chitosan or chitin nanoparticles as nanofillers not only improves the physicochemical properties but also endows the composite films with antimicrobial functions and thus broadens the application fields of biopolymer composite materials (Hosseini et al., 2015; Salaberria, Diaz, Labidi, & Fernandes, 2015). Chitin nano-whiskers (CNWs), because of their unique chemical and physical properties and pronounced antibacterial activity, provide one of the most cost-effective alternatives for the development of new antibacterial agents in applications in food packaging, cosmetics, and pharmaceutical products (Zeng, He, Li, & Wang, 2012). Starch has received considerable attention as a natural thermoplastic film matrix; however, few studies have been reported on the preparation of maize starch–based films reinforced with CNWs. Chitin nano-whiskers of slender parallelepiped rods have been successfully prepared from chitin, which has been recently explored in nanotechnology applications (Mincea, Negrulescu, & Ostafe, 2012; Muzzarelli, 2011). CNWs have many excellent properties, including biodegradability, biocompatibility renewability, and antibacterial properties (Ifuku & Saimoto, 2012). Among food-borne bacteria, Escherichia coli and L. monocytogenes are observed in a wide range of food products. In addition, these bacteria are human pathogens that cause the most economically important food-borne diseases throughout the world (Elizaquível & Aznar, 2008). These bacteria are present in foods and can multiply quickly at room temperature. Consequently, the presence of these bacteria should be controlled in the food industry. Shankar, Reddy, Rhim, and Kim (2015) reported that carrageenan/CNW nanocomposite films showed strong antibacterial activity against a Gram-positive food-borne pathogen, Listeria monocytogenes, after CNWs were added. To the best of our knowledge, reports on the reinforcement of maize starch–based biopolymer films with CNWs are not available in the literature. The objective of this study was to develop antibacterial composite films based on maize starch/CNWs and to determine the microstructures, crystalline structures, and thermal, mechanical, and barrier properties by using transmission electron micrograph (TEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). In addition, the in vitro antibacterial activities of nanocomposite films were evaluated against L. monocytogenes and Gram-negative E. 3

coli bacteria. The results of this research on antibacterial, renewable, and biodegradable CNWS starch nanocomposite films can contribute to their application in the food industry or medical fields. 2. Materials and methods 2.1. Materials Normal maize starch (with an amylose content of approximately 26.33%) was obtained from Zhucheng Xingmao Corn Development Co., Ltd. (Shandong, China). Chitin was supplied by Zhejiang Aoxing Biotechnology Co., Ltd. (Zhejiang, China) with a degree of N-acetylation of 0.96, as determined with elemental analysis. Glycerol was supplied by Tianjin Jiangtian Chemical Co. Ltd. (Tianjin, China). All other chemicals used in the present study were of analytical grade. 2.2. Preparation of CNWs The CNWs were prepared from the original raw material of chitin from crab shells based on Nair and Dufresne’s (2003) method with minor modifications. Chitin (30 g) were dissolved in 900 ml H2SO4 solution (3 mol L–1) and then incubated at 95 °C for 12 h under vigorous stirring. After acid hydrolysis, the suspension was diluted with deionized water and followed by centrifugation (5000 ×g for 15 min). This process was repeated three times with deionized water until neutral. Subsequently, the nano-whiskers were dried by lyophilization for 48 h to obtain the dried CNWs. 2.3. Preparation of the films Maize starch and maize starch/CNW composite films were prepared using a solution casting method (Sun, Xi, Li, & Xiong, 2014), with some modifications. Briefly, 7.0 g of maize starch and 3.0 g of plasticizer (glycerol) were added to 100 mL of deionized water to form starch–plasticizer slurries. Each dispersion was thoroughly stirred for 30 min in a thermostatic water bath at boiling temperature and then cooled to 50 °C. Aliquots of CNWs (0, 0.5, 1.0, 2.0, and 5.0 wt%, based on maize starch) were added to 50 mL of deionized water and then treated with an ultrasonic wave (KQ-400KDE, Kun-Shan Ultrasonic instrument Co., Ltd., Jiangsu, China) at 100 W for 10 min to ensure uniform suspension. After the CNWs were introduced, the suspensions were stirred for an additional 30 min at 300 rpm. Then, the film-forming dispersions were degassed under a vacuum (0.1 MPa) for 15 min, and the samples (about 65 g) were spread evenly over Petri dishes (15 cm

4

diameter) and dried for more than 8 h at 45 °C. All dried starch films were preserved in a relative humidity (RH = 53%, 25 °C) chamber for further testing. 2.4. Characterization and properties testing of the films 2.4.1. Transmission electron micrograph Transmission electron micrographs (TEM) were obtained with a Transmission electron microscope (TEM, HT-7700, Hitachi Instruments Ltd., Tokyo, Japan) at an accelerating voltage of 80 kV. A droplet of film-forming dispersion was placed on a carbon-coated copper grid and then freeze dried. 2.4.2. X-ray diffraction of the films The crystalline structure of the films was analyzed using an X-ray diffractometer (Bruker D8 ADVANCE, Karlsruhe, Germany). The instrument employed nickel-filtered Cu Ka radiation (k = 0.15406 nm) at 36 kV and 20 mA. The diffractograms were recorded over an angular range (2θ) of 3–40°, with a step size of 0.02° and a step rate of 2 s per step. The crystallinity of the samples was determined by plotting the peaks’ baseline on the diffractogram and calculating the area using the software spectrum viewer (Version 2.6) according to Jivan, Madadlou, and Yarmand’s (2013) method. The relative crystallinity degree was determined by the ratio of the crystalline area to the total cure area: Relative crystallinity (%) = Area under the peaks / Total curve area × 100

(1)

2.4.3. Mechanical properties A TA.XTplus texture analyzer (Lloyd Instruments, London, England) was used to determine the film’s tensile strength (TS) and elongation at break (E%). The film specimens were tested as suggested by Mehyar, Al-Ismail, Han, and Chee (2012), with minor modifications, and the tests were carried out according to the ASTM D828-97 standard test methods (ASTM, 1997). The composite films were cut into strips (1 × 10 cm), and then preconditioned at 75% (RH) for 48 h inside a sealed desiccator containing saturated sodium chloride solution at room temperature (25 ± 1 °C). The films were loaded into the testing system, which was set at an initial sample length and a grip speed of 2 cm and 100 mm min–1, respectively. 2.4.4. Thickness measurement The thickness of the films was determined using a digital micrometer (Vernier, Ningbo, 5

China, 0.001 mm accuracy) taking measurements at different positions on the film. 2.4.5. Water vapor permeability of the films Before testing, the films were conditioned at 25 °C for 48 h in a desiccator with a relative humidity (RH) of 67%. The gravimetric method was used to determine the water vapor permeability (WVP) of the maize starch/CNW composite films, and the tests were carried out according to the ASTM E96-00 (ASTM, 2000) methodology. Circular film samples were placed over the mouth of the test cup and sealed by melted paraffin in the desiccator. The test cup was about 10 mm in diameter. Anhydrous calcium chloride (0% RH) was placed inside the test cup, while a saturated sodium chloride solution (67% RH) was placed in the desiccator. The change in the weight of the cups was measured every 12 h over 2 days. The WVP was calculated as follows: WVP = (m × d) / (A × t × P)

(2)

where d is the film thickness (m), m is the weight increment of the cup (g), A is the area exposed (m2), t is the time lag for permeation (h), and P is the water vapor partial pressure difference across the film (Pa). 2.4.6. Swelling degree The swelling degree (SD) of the films was determined according to Shahzad et al.’s (2015) method. Film pieces (20 × 20 mm) were dried at 70 °C for 24 h in a vacuum ov en (Shanghai Yiheng Technology Co., Ltd., Shanghai, China) to obtain the initial dry mas s (M1). Then, the films were placed in 50 mL beakers containing 30 mL distilled water a t room temperature to allow them to absorb water, and thereby achieve a constant mass M2 before drying. Swelling degree was calculated by using the following equations: Swelling degree (%) = (M2 – M1) / M1 × 100

(3)

2.4.7. Optical and color measurement The transparency of the films was determined by measuring their light absorption at a wavelength of 600 nm using a UV-Visible Spectrophotometer Shimadzu 1601 PC (Tokyo, Japan), according to Shi et al.’s (2013) method. The film specimens were cut into strips (1 cm × 4 cm) and placed directly in the spectrophotometer test cell. Opacity was expressed as absorbance units per thickness unit. In addition, the film surface color was analyzed with a colorimeter (CR-400 Minolta Chroma Meter; Konica Minolta Sensing Inc., Tokyo, Japan) according to the method 6

described by Jang, Shin, and Song (2011) with some modifications. Films (50 mm × 50 mm strips) were placed onto a white standard plate, and Hunter values (L*, a* and b*) were measured. The Hunter L*, a*, and b* values for the standard plate were L* = 96.68, a* = 0.14, and b* = 1.94. For each sample, five measurements were taken at different locations. 2.4.8. Differential scanning calorimetry and thermogravimetric analysis The film properties were determined by differential scanning calorimetry (DSC) using a DSC1 (METTLER). Approximately 3 mg of film were weighted in hermetic pans and an empty hermetic pan was used as a reference. Samples were heated from 25 °C to 250 °C at 10 °C min–1. The thermogravimetric data (TG) for the various films was measured at a heating rate of 5 °C min–1 from 40 °C to 600 °C. 2.4.9. Antibacterial activity Antibacterial activities of neat maize starch and maize starch/CNW composite films were examined as inhibitory effects against the growth of Gram-positive bacteria, L. monocytogenes, and Gram-negative bacteria, Escherichia coli. To study the antibacterial activities, changes in the growth of L. monocytogenes and E. coli incubated in the broth medium were investigated following Li, Xing, Jiang, Ding, and Li’s (2009) and Shankar et al.’s (2015) methods. All strains were aseptically inoculated in trypticase soy broth (TSB) and brain heart infusion (BHI) broth and subsequently incubated at 37 °C for 16 h. An inoculum (100 μL) of L. monocytogenes and E. coli were aseptically transferred to 50 mL of TSB and BHI broth containing film samples (5 × 5 cm2) and subsequently incubated at 37 °C for 12–16 h under mild shaking. The inhibitory effect was estimated periodically by measuring the turbidity of the cultured medium at 600 nm using a spectrophotometer. 2.5. Statistical analysis The measurements of each property and antibacterial activity of the films were performed in triplicate with individually prepared film samples as the replicated experimental units, and mean values with standard deviations (SD) were reported. One-way analysis of variance (ANOVA) was performed, and the significance of each mean property value was determined (P < 0.05) with Duncan’s multiple range test using the SPSS statistical analysis computer program for Windows. 3. Results and discussion 7

3.1. TEM of the films

Fig. 1 TEM images of the surfaces of maize starch films reinforced with 0.5% (A), 1% (B), 2% (C), and 5% (D) of chitin nano-whiskers (CNWs); scale bars = 1 μm.

The TEM images of the maize starch film dispersions and the maize starch/CNW nanocomposite film dispersions are shown in Fig. 1. The nanocomposite suspensions with different CNW content were composed of individual and aggregated nanocrystals in a lump state. The individual CNWs present in the maize starch films (Fig. 1) had a needle-like morphology with a broad distribution in length ranging from 100 to 400 nm and lateral diameters in the range of 10−50 nm. The CNW suspension exhibited colloidal behavior, due to the protonation of the amino groups (NH3+), which induces positive charges on the surface of the crystallites and promotes the stability of the suspension as described by Watthanaphanit, Supaphol, Tamura, Tokura, and Rujiravanit (2008). As the CNW content increased, a significant tendency of agglomeration was also observed on the TEM. Similarly, Wijesena et al. (2015) found that the small chitosan nanofibers aggregated to make larger nanofiber bundles were due to the higher content. 3.2. X-ray diffraction of the films

8

F (19.4) E (17.1) D (13.8) C (11.6)

B (8.9) A (78.9) 5

10

15

20

25

30

35

40

2  () Fig. 2 X-ray diffraction patterns of chitin nano-whiskers (CNWs) (A) and maize starch films reinforced with 0% (B), 0.5% (C), 1% (D), 2% (E), and 5% (F) of CNWs; values in the parenthesis represent relative crystallinity.

The X-ray diffraction patterns of the CNWs and maize starch films reinforced with 0%, 0.5%, 1%, 2%, and 5% of CNWs are shown in Fig. 2. The CNWs diffractogram showed well-defined peaks at Bragg angles (2θ) of 9.2°, 19.1°, 23.2°, and 26.1°, typical of a highly crystalline structure, consistent with the reported values for CNWs (Chang et al., 2010b). The X-ray diffraction patterns of the maize starch/CNW films displayed characteristic diffraction of thermoplastic starch (B + V-type, 2θ of 5.4°, 16.8°, 19.7°, and 22.4°, respectively; Gironès et al., 2012). Meanwhile, Shi et al. (2006) found the formation of the B-type structure was quicker in glycerol-plasticized thermoplastic starch with less glycerol content than that with high glycerol content, and the glycerol gave the obvious diffraction peak at around 2θ values of 13.5° and 20.88°, respectively. As can be seen from this figure, the relative crystallinity of the nanocomposite films increased with the CNW content increase (0% to 5%). Moreover, the introduction of higher CNW content (5%) significantly increased the magnitude of the typical diffraction peaks of the CNWs (2θ of 9.2°, 19.1°) and the relative crystallinity of the nanocomposite film was 19.4%. These results suggested that the increase in the crystallinity of the maize starch/CNW nanocomposite films was due to the higher concentration of crystalline CNWs in the polymer matrix. 3.3. Mechanical and water barrier properties of the films

9

Table 1 Mechanical and water barrier properties parameter of maize starch (MS) films with 0%, 0.5%, 1%, 2% and 5% chitin nano-whiskers (CNWs). Sample

Thickness / mm

TS / MPa

a

WVP / 10-12 g

E/

-1 -1

%

m s Pa

176±8.65ab

4.81±0.16b

96.59±2.33b

3.69±0.07a

179±7.07a

3.57±0.17c

94.49±0.86b

0.146±0.00a

3.17±0.08b

160±8.49cd

2.22±0.11d

83.38±1.48c

0.145±0.00a

2.37±0.04d

111±4.24e

2.54±0.12d

82.15±0.78c

0.148±0.00

MS /0.5% CNWs

0.144±0.00a

2.79±0.08c

MS /1% CNWs

0.147±0.00a

MS /2% CNWs MS /5% CNWs

175±7.07

a

% 103.11±0.49a

MS /0% CNWs

bc

SD /

5.32±0.23

1.64±0.11

e

-1

The values correspond to the mean ± standard deviation. Values within each column followed by different letters indicate significant differences (P < 0.05); TS represents tensile strength, E represents elongation at break, WVP represents water vapour permeability, SD represents swelling degree, respectively.

The thickness, tensile strength, elongation at break, water vapour permeability, and swelling degree of the maize starch films and the nanocomposite films reinforced with CNWs (0%, 0.5%, 1%, 2%, and 5%) are shown in Table 1. The incorporation of CNWs did not alter the film thickness when compared to the control sample. The thickness of the films was found to be similar between 145 and 148 μm. This could be ascribed to good interfacial interaction between CNWs and the starch matrix because of the similar polysaccharide structures of CNWs and starch. Overall, the TSs of the films that contained CNWs were significantly (P < 0.05) higher than that of the pure maize starch films. Furthermore, the TS of the composite films increased to the maximum with an increase in the filler content up to 1%; however, the TS decreased slightly with more than 1% of CNWs. Similarly, Chang et al. (2010b) also found that the TS of potato starch/chitin nanoparticle composite films increased with an increase in the filler content up to 6% and then decreased with more than 6% of chitin nanoparticles. The enhanced mechanical performance of the resulting starch-based nanocomposites (CNWs/maize starch) could be due to the formation of a rigid network of the CNWs (due to the interaction among the chitin nano-size fillers by intra- and intermolecular hydrogen bonds) and/or the mutual entanglement between the chitin nano-size fillers and the starch matrix. Nevertheless, the composite films that contained more than 1 wt% CNWs exhibited a slight decrease in TS caused by aggregation of the excess CNWs. The results were consistent with those of Agustin et al. (2013), who also found a decrease in the TS of composite films with high cellulose nanocrystal content, which may be due to the 10

agglomeration of cellulose nanocrystals or non-homogeneous dispersion of cellulose nanocrystals at high concentrations. However, no significant differences were found for the E% of the maize starch/CNW composite films with an increase in the filler content up to 2%, but the E% significantly decreased with more than 5% of CNWs. This may be caused by the rigid nature of the nanofillers (Mincea et al., 2012; Reddy & Rhim, 2014). The incorporation of CNWs restricts the motion of the maize starch matrix in terms of strong interactions between the fillers and the biopolymer matrix. These results are in good agreement with previous work related to the reinforcement of composite materials with a starch-based matrix with starch and cellulose nanoparticles (Xie, Pollet, Halley, & Averous, 2013). In food packaging, the coating or film is often required to decrease or avoid moisture transfer between the food and the neighboring atmosphere. A thermoplastic starch matrix is hydrophilic showing poor barrier properties when compared to conventional polymers. As depicted in Table 2, the maximum WVP occurred in the control film and then decreased with the addition of CNWs. The WVP of the nanocomposite films decreased from 5.32 × 10–12 to 2.22 × 10–12 g m–1s–1Pa–1 with the CNW content increasing from 0% to 2%. However, superfluous CNWs (5%) aggregated easily, which actually decreased the effective content of the CNWs and facilitated water vapor permeation. These measurements could be because the addition of CNWs introduced a tortuous path for water molecules to pass through the composites. The CNWs dispersed well in the matrix at low CNW concentrations, and thus, there were fewer paths for water molecules to pass through. In contrast, superfluous CNWs aggregated (as shown in Fig. 1D) and facilitated water vapor permeation. These measurements were well in accordance with the research on the effect of the starch nanoparticle content on the WVP for potato starch films loaded with starch nanoparticles (Chang et al., 2010a). SD defines the amount of water absorbed by films and is an important property of carbohydrate film. Biopolymer films made by carbohydrates initially swell when they absorb water and then result in the changes of their structure. Thus, examination of SD is necessary for the efficient application of biopolymer films. The higher the SD is, the worse tolerance of film for water is exhibited. As can be seen in Table 1, the SD of the pure starch film is significantly higher than that of the nanocomposite films containing CNW. For a CNWs content of 5%, the SD of the 11

films reduced significantly from 103.11% to 82.15%, which indicated that the addition of CNWs (0.5%-5%) significantly improved the SD of the nanocomposite. Undoubtedly, the water resistance of the starch matrix increases steadily with addition of CNWs into the composites. Therefore, it can be concluded that the SD of the materials is suppressed in the presence of CNWs within the starch matrix. This can be attributed to the low water uptake of CNWs themselves, or to the presence of strong hydrogen bonding interactions between fiber/fiber and fiber/starch matrix (Cao et al. 2008). 3.4. Opacity and color parameters of the films Table 2 Opacity and color parameters of maize starch (MS) films with 0%, 0.5%, 1%, 2%, and 5% chitin nano-whiskers (CNWs). Samples

Opacity

L*

a*

b*

MS /0% CNWs

1.16±0.05d

86.80±0.12a

-1.39±0.02de

0.94±0.03de

MS /0.5% CNWs

1.51±0.11c

86.92±0.17a

-1.46±0.05cd

1.08±0.10cd

MS /1% CNWs

1.98±0.11b

86.62±0.14a

-1.55±0.03ab

1.14±0.07c

MS /2% CNWs

2.72±0.00b

86.77±0.22a

-1.53±0.02bc

1.17±0.02ab

MS /5% CNWs

2.98±0.08a

86.20±0.26ab

-1.60±0.03a

1.20±0.08a

Each value is the mean of three replicates with the standard deviation. Any two means in the same column followed by the same superscript letter are not significantly (P < 0.05) different by Duncan’s multiple range test.

The opacity and color parameters L*, a*, b* of the maize starch films with CNWs (0%, 0.5%, 1%, 2%, and 5%) are shown in Table 2. The opacity of starch films is of much importance when they are used to display and present the packaged food to the consumer as food surface coatings. Transparent films are characterized with low values of opacity (Zhu et al. 2014). The data on opacity in Table 2 revealed that the maize starch and maize starch/CNW nanocomposite films were highly transparent. The increase in the CNW concentration from 0% to 5% led to a significant increase in the films’ opacity. The starch films containing 5% CNWs had the highest opacity. That may be because the CNWs were embedded in the interspaces of the starch film, which prevented light transmittance and led to higher opacity. Castillo et al. (2013) also reported that film opacity increased significantly with an increase in the talc concentration in thermoplastic 12

maize starch formulations and reduced the light transmittance of the films. The high light absorbance of the films related to desirable properties of edible films since it will improve the ability of the films to protect package contents from light and enhance the quality of the packaged food, which was in line with Gomez-Guillen, Ihlb, Bifanib, Silvab, and Montero’s (2007) results. Color determination is also integral to the appearance and the consumer acceptance of packaged products. As shown in Table 2, the control and tested films presented negative values of a* and positive values of b*, which signified the films were a greenish yellow. Contrast to the maize starch film, the maize starch film containing CNWs tended more towards green with slightly increased yellowness, as suggested by their lower a* and b* values. However, the values of a*, b* and L* did not significantly differ among the nanocomposite films with varied CNWs (0%, 0.5%, 1%, 2%, and 5%). This implied that the films exhibited the same lighter color. 3.5. Differential scanning calorimeter Table 3 Thermal properties of maize starch (MS) films with 0%, 0.5%, 1%, 2%, and 5% chitin nano-whiskers (CNWs). Sample

To/°C

Tp/°C

ΔH /J g-1

MS /0% CNWs

166.09±1.15c

200.05±2.34c

31.60±0.84c

MS /0.5%CNWs

186.86±1.67a

228.92±1.73a

67.36±1.26a

MS /1% CNWs

180. 05±2.16b

220.09±1.52b

55.12±1.03b

MS /2% CNWs

180.14±0.86b

221.23±1.43b

59.39±1.30ab

MS /5% CNWs

181.73±1.34b

218.54±2.16b

59.34±1.50d

The values correspond to the mean ± standard deviation. Values within each column followed by different letters indicate significant differences (P < 0.05). TO: onset temperature, TP: peak temperature, △H: gelatinization enthalpy.

The effects of CNWs on the thermal properties of nanocomposite films were evaluated with DSC analysis. Table 3 shows the thermal parameters of the maize starch films reinforced with 0%, 0.5%, 1%, 2%, and 5% of CNWs. Compared to the pure maize starch films, a higher onset temperature (To) and peak temperature (Tp) of the maize starch films with CNWs were observed. In addition, the addition of CNWs increased the enthalpy of the composite films. These results 13

may be due to the interactions between the CNWs and the chain segments of the maize starch, which increased the crystallinity of the film. Therefore, it could be deduced that the higher gelatinization enthalpy (ΔH), the higher the compatibility of the maize starch and the CNWs. This finding is in agreement with the results reported by Suárez et al. (2013), who also observed that the addition of cellulose nanoparticles improved the ΔH and Tm of composite films. Similarly, Piyada, Waranyou, and Thawien (2013) found that the ΔH and Tm of rice starch film increased gradually as the increase of starch nanocrystals contents, which was due to the concomitant increase in the crystallinity of the starch matrix with filler content. 3.6. Thermogravimetric analysis

A

80

Mass loss rate (%/ min)

Weight loss / %

100

MC/0% CNWs MC/0.5% CNWs MC/1% CNWs MC/2% CNWs MC/5% CNWs

60

40

20

0

1.0

B

0.5

MC/0% CNWs MC/0.5% CNWs MC/1% CNWs MC/2% CNWs MC/5% CNWs

0.0

-0.5

-1.0 100

200

300

400

500

250

300

350

400

Temperature / C

Temperature / C

Fig. 3 Thermogravimetric analysis (A) and derivative thermogravimetry curves (B) for maize starch (MS) films reinforced with 0%, 0.5%, 1%, 2%, and 5% of chitin nano-whiskers (CNWs). Thermogravimetric analysis (TGA) of maize starch and maize starch/CNW nanocomposite films was carried out to evaluate their thermal stabilities and degradation profiles (Fig. 3). The TGA results exhibited a three-step thermal degradation pattern for the maize starch films and the maize starch/CNW composite films. The maize starch and nanocomposite films showed two mass losses, at around 100 °C and 200 °C, related to the volatilization of water and glycerol, respectively (Bodirlau, Teaca, & Spiridon, 2013; Teixeira et al., 2009). The main mass loss took place between 250 °C and 330 °C, which was ascribed to the decomposition of the polymeric films. Meanwhile, Fig. 3B displays the effect of the CNW contents on the maximum degradation temperature of the maize starch/CNW nanocomposite films. The maize starch film exhibited the maximum degradation temperature at 322 °C, whereas the nanocomposite film ranged from 14

309 °C to 315 °C, suggesting that the thermal stability of the maize starch film was the highest among all samples tested, and the addition of the CNWs led to the decreased thermal stability of the nanocomposite films. The main decrease in thermal stability could be due to worsening in the flexibility of the amylopectin chains of the starch in the presence of the crystalline CNWs. These results are in good agreement with previous work related to the reinforcement of the thermoplastic starch matrix with cellulose nano-size fillers (Anglès & Dufresne, 2000; Kaushik, Singh, & Verma, 2010). 3.7. Antibacterial activity of the films

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Fig. 4 Antimicrobial activity of the maize starch (MS) films reinforced with 0%, 0.5%, 1%, 2%, and 5% of chitin nano-whiskers (CNWs) against (A) L. monocytogenes and (B) E. coli. The antimicrobial activity of the maize starch and maize starch/CNW nanocomposite films against L. monocytogenes and E. coli was evaluated by measuring the optical density (OD, absorbance at 600 nm) of culture medium including the test films and microorganisms, and the results are shown in Fig. 4. Since the ODs of the medium increased with the growth of microorganisms, lower absorbance at 600 nm indicated higher antibacterial activity of the test material (Zhong, Song, & Li, 2011). As depicted in the figure, the pure maize starch films did not show any antimicrobial activity against the organisms L. monocytogenes and E. coli bacteria. As for L. monocytogenes, it was quite clear that the OD of the control assay increased consistently during the test, indicating that an appreciable growth rate of the bacteria, but the OD was suppressed greatly in suspensions that contained CNWs. Moreover, the OD of the maize starch/CNW nanocomposite films was basically unchanged with CNW contents ranged from 2% 15

to 5% in the process, whereas the bacteria continued growing at lower CNWs content (0.5% to 1%). The results indicated that the antimicrobial activity of the nanocomposite films against L. monocytogenes depended on the CNWs content. The antimicrobial mechanism could be due to the interactions between positively charged CNWs and negatively charged bacterial cell membranes, which results in increased membrane permeability and eventually causes the rupture and leakage of intracellular material (de Azeredo, 2009). Sabaa, Mohamed, Mohamed, Khalil, and Abd El Latif (2010) reported that the positively charged amino groups in chitosan bind the anionic cell surfaces of the bacteria, disrupt at least the outer membrane of the cells, and thus control the bacteria growth. As for the E. coli after 8 h incubation, the ODs kept increasing, and the trend was very similar for each suspension. Furthermore, during the same incubation period, the ODs decreased gradually at a slight rate with increasing in the CNW contents. These results suggested that the antimicrobial activities of the maize starch/CNW nanocomposite films were weaker against the Gram-negative E. coli than the Gram-positive L. monocytogenes. In addition, E. coli had flagella and fimbriae that resulted in higher mobility and thus may have made the bacteria less easily absorbable by the composite films. Similar results were obtained by Fernandez-Saiz, Lagaron, and Ocio (2009) and Takahashi, Imai, Suzuki, and Sawai (2008). 4. Conclusion In this paper, CNWs were used as the reinforcement filler for the preparation of maize starch/CNW nanocomposite films. Compared to the pure maize starch film, the nanocomposite films exhibited improved thermal properties and mechanical strength, and conferred high antibacterial activity. Furthermore, the WVP of the nanocomposite films decreased from 5.32 × 10–12 to 2.22 × 10–12 g m–1s–1Pa–1 with the CNW contents increasing from 0% to 2%. Importantly, the antimicrobial activity of the CNWs that included maize starch films were enhanced with increased CNWs content, and the nanocomposite films exhibited stronger antimicrobial activity against Gram-positive L. monocytogenes than against Gram-negative E. coli. The results showed high potential for developing antimicrobial starch-based films with the improved properties to prolong the shelf life of packaged foods.

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