Food Packaging and Shelf Life 17 (2018) 73–79
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Physicochemical, antimicrobial and antioxidant properties of chitosan/TEMPO biocomposite packaging films Bhawna Sonia, Barakat Mahmoudb, Sam Changb, Emad M. El-Giarc, El Barbary Hassana,
T ⁎
a
Department of Sustainable Bioproducts, Mississippi State University, Box 9820, Mississippi State, MS, 39762, United States Department of Food Science, Nutrition & Health Promotion Mississippi State University, 3411 Frederic St., Pascagoula, MS, 39567, United States c School of Sciences, University of Louisiana at Monroe, Monroe, LA, 71209, United States b
A R T I C LE I N FO
A B S T R A C T
Keywords: Chitosan Cellulose nanofibers Thermal stability Bacteria Packaging materials
Chitosan-based biocomposite films have attracted considerable attention due to their versatile physicochemical properties, and higher antimicrobial and antioxidant activities. This study investigated the physiochemical properties and examined the antimicrobial and antioxidant activities of chitosan/TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical) bio-composite films. The films were prepared by incorporating different ratios of chitosan (100, 85, and 75 wt.%), TEMPO cellulose nanofibers (0, 15, and 25 wt.%) and sorbitol (25 wt.%). The bio-composite films were casted in an oven at 40 °C for 2–4 days. The successful incorporation of chitosan was confirmed by Fourier transform Infrared spectroscopy (FTIR) and thermal stability (TGA) measurements. The antimicrobial activity results showed a significant reduction in the growth of Salmonella enterica, E. coli O157:H7, and Listeria monocytogenes bacteria on the surface of the films with the proportional increase of chitosan. Results also showed a significant increase in the antioxidant activity of films with high chitosan concentration. The improved antimicrobial and antioxidant activities indicate that such films can be used successfully as packaging materials for several foods.
1. Introduction
antimicrobial and antioxidant packaging films are gaining much attention from food industries for their potential applications in a variety of products including poultry, meat, cereals, cheese, fruits, and vegetables (Cha & Chinnan, 2004; Han, 2003; Shiekh, Malik, Al-Thabaiti, & Shiekh, 2013). Chitosan is a linear polysaccharide consisting of (1,4)-linked 2amino-deoxy-β-d-glucan, which is the second most abundant polysaccharide found in nature after cellulose (Dutta, Dutta, & Tripathi, 2004). Development of active biodegradable packaging films in order to improve the nutritional stability, quality and extend the storage life of food is a potential utilization of chitosan biopolymer (Chen, Zheng, Wang, Lee, & Park, 2002). Chitosan possesses strong antimicrobial, antioxidant and antifungal activities as reported by several researchers (Dutta, Tripathi, Mehrotra, & Dutta, 2009). In spite of these unique properties and numerous advantages of chitosan, its films exhibit poor mechanical functionalities as well as a weak barrier against water vapor and gases, which limits its unexclusive applications in food packaging fields. Therefore, multiple approaches have been applied to improve the barrier and mechanical performance of chitosan based films (Soni et al., 2016). In recent years, a substantial amount of research that deals with the blending of chitosan with various natural biopolymers, such as
Nowadays, packaging material is one of the major challenges that modern food industries are facing. High-quality food without chemical preservatives is the prime demand of consumers. Food packaging material with antimicrobial and antioxidant properties is an emerging technology that could have a significant impact on shelf life extension and food safety. The antimicrobial and antioxidant compounds present in packaging material can serve as a carrier to keep high concentrations of preservatives on the food surfaces. Therefore, the preparation of antimicrobial packaging films with the use of natural resources is an area of increasing interest being explored (No, Meyers, Prinyawiwatkul, & Xu, 2007; Reesha, Panda, Bindu, & Varghese, 2015; Soni, Hassan, Schilling, & Mahmoud, 2016). Biobased packaging materials could improve food quality and enhance the shelf life of food products; they are intended to function as barriers against oxygen, aroma, moisture, flavor, and oil (Rhim, Hong, Park, & Ng, 2006; Wong, Camirand, & Pavlath, 1994). In addition, biopolymer based films are excellent mediums for incorporating a broad collection of additives such as antimicrobials, antioxidants, antifungal agents, colors, and other nutrients (Han, 2003; Park & Zhao, 2004). Particularly, biopolymer based
⁎
Corresponding author. E-mail address:
[email protected] (E.B. Hassan).
https://doi.org/10.1016/j.fpsl.2018.06.001 Received 5 February 2018; Received in revised form 2 May 2018; Accepted 5 June 2018 2214-2894/ © 2018 Elsevier Ltd. All rights reserved.
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particle size and finally the moisture content was determined. The grinded shrimp exoskeletons were placed in Ziploc bags and refrigerated. Chitin was separated to several treatment steps for preparation of chitosan. In the first step, the grounded shells were soaked in 4% HCl solution (1:14 w/v) at 23 °C for 40 h to remove minerals (mainly CaCO3), then the remaining chitin was filtered and washed with distilled water till neutrality. In the second step, the demineralized chitin was treated with 5% NaOH solution (1:12 w/v) at 90 °C for 24 h to eliminate proteins and sugars from crude chitin. After cooling, chitin was collected and washed with distilled water. In the third step, chitin was deacetylated by 70% NaOH solution (1:14 w/v) at 23 °C for 75 h to produce chitosan, which was washed several times with distilled water to obtain neutral pH, followed by filtration to yield creamy white product. Finally, the moisture content was determined and the dried chitosan was kept in Ziploc bags for characterization.
starch (Bourtoom & Chinnan, 2008), cellulose (Shih, Shieh, & Twu, 2009; Velásquez-Cock et al., 2014), and several other cellulosic derivatives (Abou-Zeid et al., 2011; Dayarian, Zamani, Moheb, & Masoomi, 2014; Li, Chen, & Wang, 2015) have been reported. Polymer blending is the most efficient strategy to formulate biopolymer based films with desired properties (Soni et al., 2016). The formation of biopolymer based composite films from the utilization of green polymers is becoming an increasing acknowledged alternative for future material production for a more sustainable society. Particularly, chitosan and cellulose are primary interests due to their structural similarity which can result in materials that merge the physicochemical properties of chitosan with impressive mechanical characteristics of cellulosic fibers (Wu et al., 2004; Yin, Luo, Chen, & Khutoryanskiy, 2006). Cellulose is the most common polysaccharide on earth and a classic example of a renewable resource, which can be used to generate potential reinforcing biomaterials called cellulose nanofibers. In the last several decades, the advancement of cellulose nanofibers (CNFs) has gained considerable attention due to their low cost, low density, renewability, and nonabrasive nature. All these important characteristics of CNFs allow them to create bio-composites with easy process ability and make them attractive candidates in the nanomaterial research fields. In our previous study, transparent bio-nanocomposite films based on chitosan and TEMPO-oxidized cellulose nanofibers with enhanced mechanical and barrier properties were successfully prepared (Soni et al., 2016). These completely individualized cellulose nanofibers (CNFs) have been extracted from cotton stalks (Soni, Hassan, & Mahmoud, 2015) by TEMPO-mediated oxidation (2,2,6,6-tetramethylpiperidine-1-oxyl radical) under moderate aqueous condition (Saito, Kimura, Nishiyama, & Isogai, 2007). This oxidation phenomenon coupled with ultrasonication process selectively converts primary alcohols (eOH) to aldehyde (eCHO) and finally carboxylate groups (eCOO) (Soni et al., 2015, 2016). The width and length of TEMPO-CNFs was in range from 3–15 nm and 10–100 nm. This study aims to further examine the physicochemical characteristics of chitosan/TEMPO-CNFs films, and to examine their antimicrobial and antioxidant properties. The antimicrobial activity of these films was demonstrated against a Gram-positive bacterium (Listeria. monocytogenes) and a Gram-negative bacterium (Escherichia coli and Salmonella. enterica). The antioxidant activity was determined using DPPH and ABTS scavenging analytical assays.
2.3. Characterization of chitosan 2.3.1. Moisture content determination Moisture content of prepared chitosan was performed according to NREL/TP-510-42621 method. The water mass was determined by drying the sample to constant weight and measuring the sample after and before drying. The water weight was the difference between the weights of the wet and oven dry samples. 2.3.2. Ash content To determine the ash content value of chitosan, 2.0 g of chitosan sample was placed into previously ignited, cooled, and tarred crucible. The samples were heated in muffle furnace preheated to 700 °C for 4 h. the crucible was allowed to cool in the furnace to less than 200 °C and then placed into desiccator. Ash content was determined according to NREL/TP-510-42622 procedure. Percentage of ash value is calculated using the following equation: 2.3.3. Degree of deacetylation (%) by potentiometric titration Chitosan (0.25 g) was dissolved in 30 mL of 0.1 M HCl and diluted with 10 mL of deionized water. Under continuous stirring, 0.1 M NaOH was added dropwise until the pH reached a value of 3. A value of f(x) of the corresponding volume of NaOH added was calculated using the following formula:
V + V⎞ f (x ) = ⎛ 0 × ([H+]−[OH−]) ⎝ NB ⎠
2. Material and methods
⎜
2.1. Materials
⎟
(1)
Where V0 is the volume of chitosan solution (mL), V is the volume of NaOH added (mL), NB is the concentration of NaOH (M), [H+] is the concentration of H+ (M), [OH−] is the concentration of OH− (M). A linear titration curve was obtained by plotting f(x) vs. corresponding volume of NaOH. By extrapolating the linear titration curve to the xaxis, the volume of NaOH at the end point can be estimated. Five replicates were performed for synthesized chitosan sample. The degree of deacetylation (DD) was calculated using the following formula (Tan, Khor, Tan, & Wong, 1998)
Cellulose was isolated from cotton stalks by alkaline-acid pretreatment and used for the preparation of nanocellulose by the TEMPOmediated oxidation method (Soni et al., 2015). Chitosan (DD ∼72%) was synthesized from Mississippi gulf brown shrimp (penaeus aztucus) exoskeletons. All chemicals used in this study were purchased from commercial resources and used as received without any further purification. The following chemicals were purchased from Fisher Scientific, USA. Sorbitol, glacial acetic acid, ethanol (95%), hydrochloric acid, sodium hydroxide, potassium hydroxide, potassium persulfate, acetic acid, and TEMPO [(2,2,6,6-tetramethylpiperidin-1-yl) oxy radical]. ABTS [2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)], DPPH (2,2-Diphenyl-1-picrylhydrazyl), and Trolox were purchased from sigma Aldrich, USA. Teflon petri dish (140 mm × 140 mm) liner was purchased from Fluoro Lab, USA. Bacterial strains (ATCC), yeast extract and peptone water from Becton Dickinson, MD, USA.
DD (%) =
ɸ=
ɸ (W − 161 × ɸ) 204
× 100 +ɸ
(NA × VA−NB × VB ) 1000
(2) (3)
where NA is the concentration of HCl (M), VA is the volume of HCl (mL), NB is the concentration of NaOH (M), VB is the volume of NaOH at the end point (mL), and W is the sample mass (g).
2.2. Preparation and characterization of chitosan 2.3.4. Degree of deacetylation (%) by elemental analysis The degree of deacetylation value of chitosan samples was calculated from the following formula (Jiang, Chen, & Zhong, 2003; Kasaai, Arul, & Charlet, 2000):
Shrimp exoskeletons were obtained from Mississippi Gulf Coast, Biloxi, MS, which were dried in the oven at 40 °C for 48 h, grinded and screened through universal vibrator screen into 30 mesh−4 mm 74
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2.4. Characterization of chitosan/TEMPO-CNFs films
Table 1 Characterization of chitosan extracted from chitin. Property
Value
Chitosan yield (%) Solubility pH Moisture content (%) Ash content (%) Degree of deacetylation Elemental analysis (%) Carbon Hydrogen Nitrogen Oxygen
31.8 ± 0.4 2% Acetic acid 4.7 ± 0.2 10.3 ± 0.21 0.122 ± 0.02 72.1 ± 0.5
2.4.1. Fourier transform infrared analysis (FTIR) Fourier transform infrared analysis (FTIR) for chitosan, TEMPOCNFs, and their films were conducted by Thermo Scientific Nicolet iS50 FT-IR spectrometer in the operating range from 400 to 4000 cm−1. The resolution was set to 4 cm−1 and in all cases, 10 scans per sample were recorded. 2.4.2. Thermal gravimetric analysis (TGA) Thermogravimetric analysis of synthesized chitosan, TMEPO-CNFs, and their different films were carried out in duplicates, using Thermo Scientific SDT Q600 series Thermogravimetric Analyzer (TA instrument). Samples weighed between 9–11 mg in alumina cups and heated from room temperature to 700 °C at a heating rate of 10 °C/min. All experiments were performed under nitrogen atmosphere at flow rate 35 mL/min in order to prevent degradation by thermal oxidation.
42.15 ± 0.35 6.49 ± 0.13 6.51 ± 0.15 44.78 ± 0.23
Elemental analysis for chitin (C = 44.29 ± 0.27%, H = 6.82 ± 0.11%, N = 6.29 ± 0.16%, and O = 44.89 ± 0.27%).
DD (%) =
6.857−C / N 1.7143
2.5. Antimicrobial activity
(4)
2.5.1. Bacterial strains and growing conditions Three different bacteria were used in this study including: (a) a cocktail mixture of Salmonella. enterica (S. Enteritidis, S. Typhimurium and S. Newport), (b) a cocktail mixture of Escherichia. coli O157:H7 (C7927, EDL933 and 204 P), and (c) a cocktail mixture of Listeria. monocytogenes (Scott A, F5069 and LCDC 81–861). These strains were selected based on their prevalence in food, and their ability to survive in food over time (Ferreira, Wiedmann, Teixeira, & Stasiewicz, 2014; Heiman, Mody, Johnson, Griffin, & Gould, 2015; Jorquera et al., 2015; Lagaron, Ocio, & Lopez-Rubio, 2011). All bacterial strains were obtained from ATCC and from our personal culture collection. Bacterial strains were grown in tryptic soy broth with 0.6% yeast extract and incubated at 37 °C for 24 h prior to use. Three strains of each bacterium were mixed, with an equal volume, to give approximately 105 CFUml−1 in 0.1% peptone water.
Where C/N is the carbon/nitrogen ration measured from the elemental composition of the chitosan samples.
2.3.5. Elemental analysis Carbon, hydrogen, nitrogen content and oxygen (by subtraction) were measured with a CE-440 Elemental Analyzer (Exeter Analytical, North Chelmsford, MA). About 1–2.5 mg samples (chitin and chitosan) were weighed into a tin capsule by using an ultra-microbalance (Sartorius, Data Weighing Systems, Inc., Elk Grove, IL). The tin capsule was sealed immediately with an Exeter Analytical Capsule Sealer and placed inside a nickel sleeve for sample injection. Static combustion was conducted at 900 °C in pure oxygen, and the sample was reduced at 700 °C. Helium was used to carry the combustion products through the analytical system and also for purging the instrument. A calibration verification standard was injected after every ten samples runs to ensure quality control. Oxygen content was calculated by subtraction. All the determinations were done in triplicate (Table 1).
2.5.2. Evaluation of antimicrobial activity in vitro The antimicrobial activity of the chitosan film against Salmonella enterica, E. coli O157:H7, and Listeria monocytogenes was evaluated based on the disc diffusion assay. 100 μl of each strain mixture was streaked onto the appropriate selective medium: xylose lysine desoxycholate (XLD) (Difco, Becton Dickinson) for Salmonella. enterica, cefixime-tellurite sorbital MacConkey (CT-SMAC) (Difco, Becton Dickinson) agar for E. coli O157:H7, and modified oxford agar (MOA; using DIFCO Modified Oxford Antimicrobic Supplement) for Listeria. monocytogenes. Chitosan/TEMPO-CNFs film samples (100/0, 85/15, 75/25) and control sample (TEMPO-CNFs film) were prepared in discs of 8 mm in diameter and sterilized using 1 kGy X-ray irradiation. The film samples were placed on the surface of inoculated agar plates by using a sterile tweezers and gently pressed to ensure full contact to agar surface. The plates were then incubated at 37 °C for 24 h, then the zone where the film was placed and its surroundings were carefully observed (Rhim et al., 2006). All experiments were repeated three times using two samples per experiment for a total of six data points per treatment. The inhibitory activity was measured based on the diameter of the clear inhibition zone. If there was no clear zone surrounding, it was assumed that there was no inhibitory zone. Contact area was used to evaluate growth inhibition underneath film discs in direct contact with target microorganisms in agar.
2.3.6. Casting of chitosan/TEMPO-CNFs films Films were casted using the same method described in the previous study (Soni et al., 2016). Vacuum was applied to degas the film solutions to prevent microbubble formation in the films. Solutions were poured onto 140 mm × 140 mm petri dish liners and dried in oven at 40 °C for 2–4 days. It was then peeled carefully from the petri dish. Before characterization, all film samples were preconditioned for at least 48 h in a constant temperature and humidity chamber at 25 °C and 50% relative humidity to ensure the stabilization of their water content. Water vapor permeability, oxygen permeability, and tensile strength were studied in our previous work. The ratio of film components, thickness, and the moisture content value for each film are shown in Table 2. Table 2 Various compositions of chitosan/TEMPO-CNFs biocomposite films (Sorbitol = 25 wt%, 2% acetic acid solution = 1.25/150 g/mL, and casting solution = 100 mL). Samples (wt/ wt)
Chitosan (wt %)
TEMPO CNFs (wt%)
Thickness (μm)
100/0 85/15 75/25 TEMPO-CNFs
100 85 75 0
0 15 25 100
114.1 129.4 149.7 118.6
± ± ± ±
Moisture content (wt%) 0.4 0.3 0.5 0.4
2.6. Antioxidant property
6.6 ± 0.21 7.7 ± 0.24 10.2 ± 0.20 8.1 ± 0.26
2.6.1. Sample preparation To obtain the sample extracts, 250 mg of synthesized chitosan and each chitosan/TEMPO-CNFs based bio-composite film (100/0, 85/15, and 75/25) were placed in a capped plastic centrifuge tubes and 25 mL 75
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3348 cm−1is associated with OeH and NeH hydrogen bonds. The dominant spectral band at 3340 and 1040 cm−1 corresponds to stretching vibrations of OeH and CeO ether groups. The band at 1592 cm−1 is associated with NeH stretching and bending (El Miri et al., 2015). The spectral bands observed in all bio-composite films in the region of 1639–1648 cm−1are attributed to OeH bending of adsorbed water. The spectra were developed after drying, but some of the water adsorbed by the polymers is difficult to extract due to the polymerwater interaction (Morán et al., 2008). The peaks in the region of 2898–2900 cm−1 indicate antisymmetric and symmetric vibrations of CH2 groups (Soni et al., 2015). Other peaks include CH2 scissoring at 1410–1420 cm−1, CeH bending at 368–1373 cm−1, CH2 wagging at ∼1317 cm−1, ∼CeOeC pyranose ring stretching vibration at 1048 cm−1 in both chitosan and cellulose molecules, and β-glycosidic linkages at 891–896 cm−1 (Chen et al., 2011; Garside & Wyeth, 2003; Kumar, Negi, Choudhary, & Bhardwaj, 2014). The major bands of chitosan at 1633 cm−1 and 1520 cm−1 corresponds to the carbonyl (C]O) stretching of acetyl groups and NeH bending vibrations (amide and amine groups). The broad band at 3200–3450 cm−1 in the bio-composite films moved slightly to higher wavenumbers, which suggests a strong interaction between positively charged ammonium groups of chitosan (eNH3+) and the negatively charged carboxylate groups (eCOO) on the TEMPO-CNFs (de Mesquita, Donnici, & Pereira, 2010; Li, Zhou, & Zhang, 2009). When TEMPO-CNFs were included in chitosan films, the band corresponding to NeH bending almost disappeared, which suggests the formation of hydrogen bonds between chitosan and CNFs. These results indicate that intermolecular hydrogen bonding occurs between TEMPO-CNFs and chitosan molecules in the blended films, leading to a good miscibility of the blends (Soni et al., 2016).
of methanol were added. The tubes were then centrifuged (Fisher Scientific, accuSpin™ 3R) at 1000 rpm for 10 min at 30 °C and the supernatant liquid was transferred to another set of glass tubes. The extracts obtained were stored at 23 °C and measured before 24 h. 2.6.2. DPPH free radical scavenging activity Antioxidant activity of chitosan and chitosan/TEMPO-CNFs biocomposite films was evaluated by using DPPH free radical scavenging assay according to the method described by (Wangcharoen & Morasuk, 2007) with slight modifications. An aliquot of 0.6 mL supernatant (prepared samples), 1.2 mL 0.8 mM DPPH solution and 10.2 mL methanol were mixed and kept at room temperature for 1 min. Then, the absorbance at 517 nm was measured using a Varian carry100 UV–Vis spectrophotometer (Australia). The DPPH scavenging activity was calculated against a calibration curve established with Trolox and expressed as mg Trolox equivalent/g film. Two replicate runs were performed for each experiment. 2.6.3. ABTS free radical scavenging activity ABTS free radical scavenging activity of bio-composite films and synthesized chitosan was measured following the method described by (Carrasco-Castilla et al., 2012) with modifications. ABTS radical cation (7 mM final concentration) was prepared by dissolving ABTS in 2.45 mM potassium persulfate and kept in a dark place for 12–18 h. The absorbance of this radical cation solution was adjusted to 0.70 ± 0.02 at 734 nm with methanol dilution. This solution was used as a working solution. A 5.0 mL dilute solution of ABTS radical cation and an aliquot of 0.05 mL supernatant of each sample were mixed and allowed to proceed for 6 min. The absorbance values for all solutions were measured at 743 nm. The activity was determined as mg Trolox equivalent/ g sample by calculating against a calibration curve established with Trolox. Two replicate runs were performed for each experiment to minimize the experimental error.
3.2. Thermal gravimetric analysis (TGA) The thermal stability of prepared chitosan, TEMPO-CNFs, and all bio-composite films of different composition is presented in Fig. 2. All samples displayed a slight weight loss at low temperature from 100 to 125 °C in range due to evaporation of absorbed and intermolecular Hbonded water and acetic acid (Kumar et al., 2014; Soni et al., 2016). However, the pyrolysis processes and degradation behaviors of these samples are completely different in the high temperature range. The TGA curve represented by weight (%) and solid lines, where thermal degradation of chitosan initiated at 292 °C in nitrogen atmosphere. DTG curve represented by deriv. weight (%/°C) and dashed lines. An extended pyrolysis process starts at 215 °C and reaches a maximum at 380 °C (Neto et al., 2005; Tirkistani, 1998). This range corresponds to the decomposition (oxidative and thermal) of biopolymers, and vaporization and elimination of volatile products. The thermal degradation of TEMPO-CNFs begins at 215 °C and the entire pyrolysis process of TEMPO-CNFs are in temperature range of 180–260 °C and 260–350 °C, thus showing two broad peaks. The reason may be the formation of carboxylate groups (eCOO) in TEMPO-CNFs by TEMPO-mediated oxidation at C6 primary eOH groups of cellulose that lead to a significant decrease in the thermal degradation point (Soni et al., 2016). This result reveals that created carboxylate groups through TEMPO-mediated oxidation contributes to a significant reduction in degradation temperature due to their nano-size fibers (Soni et al., 2015). In bio-composite films, the amount of absorbed water in pure chitosan (100/0) film is higher than that of blended chitosan/TEMPOCNFs film (specially, 75/25), suggesting that the TEMPO-CNFs were properly dispersed within the chitosan matrix, for this reason preventing the absorption of moisture by the films (Soni et al., 2016). In TGA thermograph, thermal degradation of the 100/0 chitosan film and other TEMPO-CNFs reinforced films (85/15 and 75/25) initiated at 240 to 250 °C under N2 atmosphere. In spite of, all films in DTG curve showed prominent pyrolysis with single step degradation and the major associated weight loss is observed from 220–380 °C temperature range.
3. Results and discussion 3.1. Fourier transform infrared analysis (FTIR) Fig. 1, shows the FTIR spectrum of prepared chitosan (penaeus aztucus) species, TEMPO-CNFs and bio-composite films of different compositions. Glucosamine, a major group (eNH2 which represents the free amino) present in chitosan, shows a main absorption band between 1220 and 1020 cm−1 at C2 position. TEMPO-CNFs have a prominent peak at 1610 cm−1, which corresponds to carbonyl groups (Morán, Alvarez, Cyras, & Vázquez, 2008; Soni et al., 2015). In all bio-composite films of different compositions (100/0, 85/15, and 75/25) the band at
Fig. 1. FTIR spectra of prepared chitosan, TEMPO-CNFs, and different biocomposite films. 76
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Fig. 2. TGA and DTG curves of prepared chitosan, TEMPO-CNFs, and different biocomposite films.
effect of pH on microorganisms must be considered together with the effect of chitosan. Therefore, the combined effect of the chitosan and pH together is possibly the most evident explanation of the antimicrobial effect of chitosan (Alishahi & Aïder, 2012; Lagaron et al., 2011). More recently, it showed that chitosan is more powerful antibacterial agent against Gram-negative bacteria (E. coli and Salmonella. enterica) than against Gram-positive microorganisms (Listeria. monocytogenes) (Kong et al., 2010). While the results received from the antimicrobial testing on chitosan/TEMPO-CNFs films does not show any difference between both types of bacterial strains. The films made of different compositions of TEMPO-CNFs in chitosan matrix are transparent, homogeneous, flexible, and creates strong interaction and adhesion on the interfaces of both polymeric chains (Soni et al., 2016). TEMPO-CNFs allow the chitosan biopolymer to develop a continuous films of various shapes easily without any deformation (Nordqvist et al., 2007). This excellent dispersion of nano-sized TEMPO-CNFs with diameters of 3–20 nm and length 10–100 nm (Soni et al., 2015) are strongly blended in chitosan biopolymers and formed a composed framework that is directly correlated with its effectiveness for enhancing the properties of bio-composite films. The higher concentration of TEMPO-CNFs (15–25 wt.%) increases the inter chain bonding between both the biopolymers which leads to obstruct the water vapor transmission and reduce water vapor diffusivity through the film. Also, films with 85/15 and 75/25 demonstrated excellent oxygen barrier properties (Soni et al., 2016). These properties improve the antimicrobial property of these bio-composite films and lead to a limitation of aerobic spore germination.
As shown in this research, there is not much difference in the degradation temperature for all bio-composite films. Therefore, these films demonstrate reasonable thermal stability, which is mainly due to the presence of the great compactness and crystalline structure between both TEMPO-CNFs and chitosan polymer (Soni et al., 2016). This result reveals that the introduced eCOOH moieties can affects the thermal stability of the chitosan based composites due to their nano-size fibers and large number of free ends. 3.3. Antimicrobial activity Antimicrobial activity is very important for food packaging, in this research it is evaluated by disc diffusion method. Fig. 3 presented photographs of the antimicrobial properties of the control sample and different bio-composite films with three cocktail mixtures of bacterial strains. All chitosan/TEMPO-CNFs films (100/0, 85/15, and 75/25) showed a clear antimicrobial property on both Gram-positive and Gram-negative bacteria. TEMPO-CNFs film (control sample) displays no antimicrobial effect, whereas the films with different chitosan/TEMPOCNFs compositions shows complete inhibition for all three kinds of bacteria (Salmonella enterica, E. coli O157:H7, and Listeria monocytogenes). Film with 100/0 composition presents great prevention and higher antimicrobial activity against all bacterial strains, while 85/15 and 75/25 film compositions showed less inhibition. This may be due to the concentration of amino groups in chitosan film (100/0) combination that concerns the antimicrobial property. Therefore, significant reduction of the growth of tested bacteria increases by increasing the concertation of chitosan percentage in the film (antimicrobial activity: 100/0 > 85/15 > 75/25). For 85/15 and 75/25 blends, TEMPO-CNFs dispersed homogeneously in the chitosan matrix. Thus, the local concentration of amino groups in these samples are smaller than that of neat chitosan film (100/0). One of the reasons for antimicrobial activity of chitosan can be its positive charge of the amine group (eNH3+) at pH values lower than the pKa (pH < 6.3) that carries 50% of its total electric charge. It allows the interactions with negatively charged microbial cell membranes, which is likely to cause a leakage of nucleic acids, proteins, low-molecular weight materials, and other intracellular constituents (Helander, Nurmiaho-Lassila, Ahvenainen, Rhoades, & Roller, 2001; Lagaron et al., 2011). Another reason for antimicrobial characteristics can be the flocculation and adsorption of electronegative substances in the microbial cell through chitosan, then disturbing the physiological activities of the microorganisms and causing their death (Kong, Chen, Xing, & Park, 2010; Raafat & Sahl, 2009). In addition, it is very important to mention that chitosan dissolves only in acidic media and the
3.4. Antioxidant activity DPPH and ABTS free radical scavenging activities of chitosan/ TEMPO-CNFs bio-composite films and control are shown in Fig. 4. In this study, Trolox, an antioxidant with a wide range of antioxidant properties, was used as a common reference standard for these two antioxidants test (Thaipong, Boonprakob, Crosby, Cisneros-Zevallos, & Byrne, 2006). Previous studies indicated that chitosan exhibited good antioxidant activities, especially the scavenging ability towards hydroxyl radicals (Yen, Yang, & Mau, 2008). It was also indicated that the degree of antioxidant activity in the bio-composite films is generally proportional with the amount of added antioxidant compounds in the film (Moradi et al., 2012). These observations seem to be true for the current prepared films because the antioxidant activity was gradually increased with incorporating more chitosan in the film. As it is clear in the figure, TEMPO-CNF film (control) showed very low radical 77
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Fig. 3. Antimicrobial property of different biocomposite films on inoculated media with: (a) a cocktail mixture of Salmonella. enterica; (b) a cocktail mixture of E. coli O157:H7; (c) a cocktail mixture of Listeria monocytogenes.
8.2, respectively. It can be concluded that the antioxidant activities of the films towards ABTS radical is slightly higher than that towards DPPH radical. These antioxidant results are in accordance with previously reported data (Genskowsky et al., 2015). The scavenging mechanism of chitosan is related to the fact that the free radical can react with the free amino (eNH2) groups to form a stable macromolecule radicals (Yen et al., 2008) and enhance the antioxidant activity. Gradual increase of TEMPO-CNFs into chitosan matrix in acidic solution converts free eNH2 groups into ammonium (eNH3+) groups and form electrostatic bond with carboxylic (eCOO) groups present on TEMPOCNFs (Soni et al., 2015). Therefore, chitosan biopolymer and its higher concentration might be primarily responsible for the antioxidant activity of the bio-composite films. 4. Conclusions Four different compositions of chitosan/TEMPO-CNFs films were developed, characterized, and their antimicrobial and antioxidant activities were tested. FTIR spectrum indicated the presence of intermolecular hydrogen bonding between TEMPO-CNFs and chitosan molecules in the blended films which leads to good miscibility of the blends. The thermal behavior revealed that the biocomposite films had higher thermal stability. Antimicrobial effects of all biocomposite films with various ratios and concentrations against food pathogenic bacteria namely Salmonella enterica, E. coli O157:H7, and Listeria monocytogenes were evaluated in vitro and confirmed by disc diffusion assay. The antimicrobial testing method demonstrate that all biocomposite films
Fig. 4. Antioxidant activities of different biocomposite films determined by two antioxidant assays (DPPH and ABTS assay).
scavenging activity for the two assays compared with chitosan/TEMPO compositions. Increasing the amount of chitosan in the films resulted in gradual increase in the antioxidant activities of the films. The antioxidant activity for the biocomposite films (75/25, 85/15, and 100/0) towards ABTS radical scavenging was 5.8, 7.9, and 11.3, respectively. Similarly, the antioxidant activity for the biocomposite films 75/25, 85/15, and 100/0 towards ABTS radical scavenging was 2.1, 6.1, and 78
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possess antimicrobial activity against food pathogenic bacteria, even though 85/15 and 75/25 films have less concentration of chitosan. All films showed the same inhibition against Gram-positive and Gram-negative bacteria. In a similar way, antioxidant activity has increased with higher concentration of chitosan in the film. Results of this study indicated clearly that incorporation of TEMPO-CNFs into a chitosan biopolymer can leads to production of new class from bionancomposite with higher antimicrobial and antioxidant activities.
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