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Development of biodegradable composite chitosan-based films incorporated with xylan and carvacrol for food packaging application
Food Packaging and Shelf Life 21 (2019) 100344
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Development of biodegradable composite chitosan-based films incorporated with xylan and carvacrol for food packaging application
T
Donatien Pascal Kamdema, , Zhu Shena, Omid Nabinejada, Zuju Shub ⁎
a b
School of Packaging, College of Agriculture and Natural Resources, Michigan State University, MI 48824, East Lansing, USA Division of Packaging Engineering, School of Light Textile Engineering and Art, Anhui Agricultural University, Hefei 230036, China
ARTICLE INFO
ABSTRACT
Keywords: Biocomposites Xylan Chitosan Mechanical properties Thermogravimetric analysis Packaging
This paper presents an experimental study on the development of uniform and homogeneous composite chitosan based flexible films containing various amount of xylan from 0 to 25 wt percent and carvacrol. The influence of the addition of xylan on the composite films was evaluated by measuring the tensile strength, moduli and elongation at break. The tensile strength and moduli of composite films containing 20 wt. % increase considerably by 73.9% and 66.8%, respectively. The elongation at break also increases considerably with the addition of xylan, the maximum value of 4.5% was obtained with film containing 8 wt.% xylan. Thermogravimetric analysis (TGA) indicates that the addition of xylan reduces the rate of degradation and shifts the thermal degradation peak from 290.7 to 276.8 °C for the film containing 20 wt.% xylan. Investigation on the antibacterial activity showed that Tetracycline reference control is effective in controlling the growth the bacteria while the incorporation of carvacrol in chitosan xylan composite is not effective.
1. Introduction Plastics derived from fossils fuels have been intensely used for consumers’ products packaging because of their low weight and affordable costs (Tharanathan, 2003). The global plastic packaging market size is projected to reach USD 400 billion by 2022 at a compound annual growth rate (CAGR) of 5% between 2016 and 2022 (HalfCooked Research Reports, 2019). However, the unpredictable price variation of petroleum fossil fuel promotes the variation of the cost of synthetic plastics making the plastic market cost uncertain, which is a non-desirable parameter for packaging manufacturers. The consumers’ awareness of the non-biodegradability, non-compostable packaging and some others negative impact of synthetic plastic packaging at the end of their service life have driven synthetic plastic manufacturers of the global plastic packaging market to find alternative raw materials. It has been reported that the cumulative production of plastics over the period of 1950 to 2015 reached 7.8 billion metric tons - more than one tone of plastic for every person alive today (Hannah, 2018). The majority of them was petroleum-based plastics with low to negligible biodegradability and disposed off yearly into landfills with generation of undesirable byproducts including methane gas. Moreover, greenhouse gas (GHG) and particulates are also released into the surrounding environment during the extraction, transportation, and manufacturing of synthetic plastic from petroleum fossils oils (Robertson, 2008). ⁎
In 2015, Environmental Protection Agency (EPA) reported that 30% of the municipal wastes -mounting to 78 million metric tons were mainly plastic-based packaging materials (Robertson, 2008) with considerable negative impacts on the environment, animal and human life such as irregular climate patterns. Several strategies and means have been proposed to mitigate these changes to sustain the planet. One of the strategies include the uses of biobased available materials or residues with low carbon fingerprint, requiring low processing or manufacturing energy and low emission of greenhouse gas which is the case of several plants and animals residues. Cellulose, hemicellulose, lignin, and starch from plant cell walls are good candidate raw materials from biomass currently used and underdevelopment to make packaging films with some limited properties. Desired packaging material should possess mechanical, optical, thermal properties to offer adequate protection against exterior factors such as vibration, tension, compressions during the logistics. Packaging should also prevent the transfer of unwanted substances such as microbial contamination, moisture, and oxygen to protect the contents (Jamal et al., 2019). They also should be recyclable; renewable, and release low to negligible GHG in the environment (Robertson, 2008). Packaging materials from biological sources including forest, agricultural, plants, animal and seafood residues are known for their biodegradability (Khan et al.2016). They are renewable, available, sometimes edible and relatively low cost and capable to generate sustainable
Corresponding author. E-mail addresses: [email protected] (D.P. Kamdem), [email protected] (Z. Shu).
https://doi.org/10.1016/j.fpsl.2019.100344 Received 17 June 2018; Received in revised form 20 May 2019; Accepted 29 May 2019 Available online 04 June 2019 2214-2894/ © 2019 Published by Elsevier Ltd.
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additional income to fishing or agriculture or forest industries. They are considered residues of another and are considered attractive alternatives to replace fossil fuels plastics packaging materials in several applications. Several researches have focused and reported on the possibility of using chitosan as raw materials for flexible films with properties compared to that of existing synthetic plastic. Chitosan is a cationic natural polymer generated from the de-acetylation of chitin, the major component of the shells of crustaceans (Shen & Kamdem, 2015b). Chitosan is a linear binary heteropolysaccharide composed of β-1,4-linked glucosamine and N-acetylglucosamine, and has been proven to be non-toxic, biodegradable and biocompatible polymer with potential antimicrobial properties (Ojagh, Rezaei, Razavi, & Hosseini, 2010). Chitosan made film is reported to be a good candidate to produce films with permeability to gases (CO2 and O2) comparable to that of commercial compostable poly lactic acid values of 1.99 × 10−17 and 0.49 × 10−17 kg m/m2 s Pa, respectively. Acceptable flexible packaging should also exhibit a tensile stress value of 75 MPa, a tensile modulus and elongation at break close of 2.5 GPa and 75% minimum to be compared to existing commercial materials (Leceta, Guerrero, & de la Caba, 2013; Selke & Culter, 2016). However, development and utilization of chitosan are currently limited because of their low stretchability of 7 to 10% (Shen & Kamdem, 2015a) and their undesirable water relationship in some applications due to the presences of numerous hydroxyl groups. The use of chitosan to produce flexible packaging is limited because of their relatively low tensile strength ranging from 60 to 75 MPa, high water absorption and high oxygen permeability (Shen & Kamdem, 2015b). High water permeability packaging materials is a challenge to protect some fresh and dried food. Water may promote the growth of biodegradable microorganisms such as mold and mildews. To improve the shelf life of the contents, the addition of antimicrobial in the chitosan film during the manufacture may help control the growth of these pathogens. The development of commercially acceptable and performant chitosan-based films will require the increase of the elongation and the reduction of their water vapor retention value to be close to that of flexible films made of synthetic plastic. Several methods have been proposed to improve the barrier and mechanical properties of chitosanbased films including the replacement or substitution of some hydroxyl groups through grafting and/or addition of hydrophobic moieties including fatty acids (Perdones, Vargas, Atarés, & Chiralt, 2014; RuizNavajas, Viuda-Martos, Sendra, Perez-Alvarez, & Fernández-López, 2013; Shen & Kamdem, 2015b). Hemicellulose based films with a tensile strength of up to 100 MPa (Shen & Kamdem, 2015a) much higher than that of some synthetic plastic such as high density polyethylene with 30 MPa tensile strength. It is worth mentioning that hemicellulose is the second most abundant polymer that can be obtained from plants and animal tissues; 15 to 25% of wood is hemicellulose. Natural essential oils such as tung oil, cedarwood oils have shown some potential antimicrobial when included in chitosan film matrix. However, the addition of a hemicellulose such as xylan, which known to have good tensile strength and low oxygen permeability, may improve the properties of composites film xylan (Gordobi, Egues, Urruzola, & labidi, 2014). Hemicellulose based films with a tensile strength of up to 100 MPa (Shen & Kamdem, 2015a) much higher than that of some synthetic plastic such as high density polyethylene with 30 MPa tensile strength. The objective of this study is, therefore, to evaluate the feasibility of manufacturing a composite flexible film for packaging using chitosan as the base polymer matrix, carvacrol to control microbial protection and various amounts of wood xylan from 0 to 25 wt% to improve mechanical properties. The hypothesis of the development of this film formulation with additive proposed will result in the increase of some mechanical and biological properties with the addition of xylan and carvacrol due to the high reactivity of the amine group of chitosan and
the hydroxyl groups from xylan and carvacrol. In addition, the thermal degradation of composite films, which is an important parameter during the manufacturing process such as melt compounding, will also be investigated to figure out the effect of these additions on the thermal stability of compounds. As a consequence of high water-permeability, chitosan film may be susceptible to some bacteria. Therefore, antibacterial activities will also be studied on the films to investigate the usefulness of these composite films for food packaging applications. 2. Materials and methods 2.1. Materials Chitosan from shrimp shells with a deacetylation degree of 75, glacial acetic acid 99%, Span 80®, high purity (98%) carvacrol, and xylan were purchased from Sigma Chemical Co. St. Louis, MO and used without further purification. In our previous study, the molecular weight distribution of chitosan used in this study was determined by gel permeation chromatography (GPC, Waters Model 500 HPLC pump, Milford, USA) with a Millennium software program in water. The number-average (Mn) and weight–average (Mw) molecular weights were 138 and 199 kDa, respectively (Shen & Kamdem, 2015a). Xylan was a light brown solid fine powder extracted from beechwood 95% pure. Carvacrol and xylan were stored in dark container in the laboratory kept at 23 °C. The bacterial strains used in this study were Listeria innocua ATCC33090 (L. innocua), and Escherichia coli ATCC25922 (E. coli). These bacteria were kept at 4 °C. Subcultures of bacteria were carried out every 7 days to maintain bacterial viability. Overnight cultures of these bacteria were grown in Brain-heart infusion (BHI) broth at 37 °C in an incubation chamber from Sheldon Manufacturing Inc, Cornelius, OR. The bacterial population in all the inoculated media was estimated at more than 1 × 107 CFU/ml after 24 h incubation. 2.2. Preparation of the film-forming dispersions Aqueous solution of chitosan was prepared by dissolving 1.5 g of chitosan powder in 148.5 ml of aqueous acetic acid solution representing a 0.5%v/v to obtain a 1%w/v chitosan solution, using a magnetic stirring plate following the method previously published (Shen & Kamdem, 2015b). The chitosan solution was stirred overnight at room temperature to achieve complete dissolution and dispersion. Xylan powder was dissolved in hot water at a solid : liquid ratio of 1:50 during 12 h using magnetic stirring. The solution of xylan in water was filtered to remove the undissolved particulates before mixing with chitosan aqueous solution. Appropriate amounts of xylan solutions were added to the chitosan solution to reach a final concentration of 10, 20, 30, and 40%w/w xylan on the basis of neat chitosan weight. Then 10% w/w of carvacrol, 1%w/w of Span 80® at a level of neat chitosan weight was added to the mixture and stirred for 30 min to achieve a stable well disperse emulsion. Table 1 shows the sample compositions in this study. Finally, emulsions were homogenized with an Ultra-Turrax (IKA, Canada) at 2500 rpm for 4 min. After cooling the resulting mixture at room temperature, it was degassed under vacuum for 5 min in order to Table 1 Sample compositions.
2
Sample ID
Chitosan/ Xylan (%)
Chitosan (g)
Xylan (g)
Carvacrol (g)
Span 80® (g)
Chitosan Xylan 8% Xylan 14% Xylan 20% Xylan 25%
0 10 20 30 40
82.5 76.1 70.8 65.8 61.8
0.0 7.7 14.0 20.0 24.8
8.3 7.6 7.1 6.6 6.2
1.0 1.0 1.0 1.0 1.0
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remove all bubbles. A composite film without xylan was also produced and considered as control. The emulsions were then spread over polystyrene petri dishes (65 mm diameter), which were placed on a leveled surface and allowed to dry for approximately 48 h at 30% RH and 23 °C. Uniform film thicknesses were achieved by casting the same amount of film-forming solution onto each plate. After drying, the films were peeled off the casting surface and maintained at 23 °C and 50% relative humidity (produced with saturated Ca(NO3)2 solution) in a conditioning desiccator until further evaluation.
for each formulation. The agar dilution test was used to determine the density of the bacteria in broth. A serial ten-fold dilutions of broth from 1 to 1 × 10−9 g/ml were made by adding 1 ml of fresh broth into 9 ml of sterile 0.9% sodium chloride solution. After shaking and completely mixing followed by serial dilutions, 1 ml of each solution was sub-cultured on agar plates and dispersed. The settled plates were sealed and placed in an incubator for 24 h. The number of colonies forming units (CFU) of bacteria that appear on the countable agar plate (between 30 and 300) was estimated. For the agar diffusion methods, the films were cut into 10 mm diameter disks with a scissor. Film cuts were placed on Brainheart infusion (BHI) agar for L. innocua and Tryptic Soy Agar (TSA) for E. coli. Films disks specimens were then placed on the surface of inoculated agar petri dish plate, stored in an incubation chamber at 37 °C for 24 h. The area of the inhibition zone defined as zone without apparent visual growth of bacteria was measured with a caliper to the nearest 0.01 mm (Aa). The whole zone area that was pre-inoculated with bacteria was measured and used as the potential surface of bacteria growth and corrected by subtracting the film surface (Ab). The tests were carried out in triplicate for each type of film. Tetracycline was used as a positive control reference well known to inhibit bacteria growth and used to establish the antimicrobial growth. The antimicrobial index was calculated as the percentage of the value of inhibited surface measured with a caliper by the total potential surface growth of inoculated petri dish. A 100% antimicrobial index corresponds to a complete surface inhibition of microbial growth on the petri dish surface while a 0% surface inhibition index is a full microbial growth on the surface of petri dish or 0 cm2 surface inhibition. The antimicrobial index growth for bacteria used in this work was computed by using Eq. (1):
2.3. Film characterization 2.3.1. Film thickness Film thickness was determined using a hand-held digital micrometer (Mitutoyo No. 293-766, Tokyo, Japan) with a precision of 0.001 mm. Measurements were carried out on at least five random locations on each film, and the mean thickness value was calculated and used to calculate the mechanical properties of the films. 2.3.2. Tensile properties of films Standard method D882-12 (ASTM, 2018) was used to measure the tensile properties of films. Films were cut into strips with a test dimension of 50 mm long and 6.35 mm wide. The composite films were determined at 23 and 50%RH with an Instron 5565 Universal Testing Machine (Instron, Canton, MA). The initial gauge length was set to 25 mm and films were stretched using a crosshead speed of 1 mm/min. A microcomputer was used to record the stress-strain curves. Tensile strength and elongation at break were calculated. Four replicates of each test sample were run to achieve dependable data. The Young’s modulus was calculated using Hooke’s law which is the ratio of the stress to the strain in tensile testing and the modulus of elasticity as the initial linear slope of the curves of stress versus strain as related to the equations below:
Antimicrobial index (%) =
Surface inhibited × 100% Total potential surface
(1)
The zero or minimum surface inhibition of bacteria was obtained by using film with no carvacrol content while the maximum inhibition surface was estimated using films containing 2% tetracycline as reference maximum antibacterial (Shen & Kamdem, 2015a).
Young's Modulus (M) = (Stress (MPa) / (Elongation,%) Where; the Young modulus is expressed in force per unit area, the stress in force per unit area and the elongation in percentage of dimension change from the initial dimension. The greater the value of stress or the modulus results in the greater the resistance to deformation or the rigidity of a material. Young modulus is a good indicator of the rigidity of a material while strain or elongation is more related to flexibility and searchability.
3. Results and discussion 3.1. Physical properties of the films 3.1.1. Effect of acetic acid concentration It has been stated that acetic acid used in this study to dissolve chitosan. In order to study the effect of various concentration of acetic acid on the tensile strength and elongation at break is presented in Fig. 1. It is clear from Fig. 1 that the specimen with 0.51% acid acetic exhibits the optimum values for the tensile strength (166.63 MPa), elongation at break (6.72%) and young modulus (6.26 GPa). Since the strength times strain corresponds to the toughness, it can be concluded that the sample with 0.51% acetic acid presents the maximum toughness compared to the other concentration. Therefore, acetic acid with 0.51% concentration has been fixed for the next steps in this study. Chitosan has been previously reported (Xia, Wu, & Chen, 2013) to be solve in 1% (or even more (Shaojie, Xuefeng, Deyong, Yiping, & Kangde, 2004)) acidic acid. The finding from this study indicates that 0.5% acidic acid is appropriate concentration to dissolve chitosan and that chitosan and xylan are miscible in slightly acidic solution to form flexible films. This is important as using less acetic acid results in more ecofriendly materials with better mechanical properties for development of a flexible film based on chitosan. The use of 0.55% at higher acetic acid to dissolve Chitosan may correspond to hydrolysis and depolymerization of chitosan with reduction of strength properties in agreement with literature on the impact of depolymerization on properties of polymers (Nunes, Martin, & Johnson, 1982).
2.3.3. Thermogravimetric analysis Thermogravimetric analysis (TGA) was performed to evaluate the thermal stabilities of composite films using a TGA 2950 running with the Universal Analysis Software Package V.3.9a (TA Instruments, New Castle, DE, USA). Samples of approximately 5 mg were heated from 50 to 650 °C at 10 °C/min heating rate under a nitrogen flow of 70 mL/min. Weight losses of samples were measured as a function of temperature. All the measurements were conducted in duplicate. 2.3.4. Antimicrobial activity The agar diffusion method was used to qualitatively evaluate the antimicrobial activity of the films according to (Pelissari, Grossmann, Yamashita, & Pineda, 2009), with modifications. The films were cut into 10 mm diameter disks with a scissor. Film cuts were placed on Brain-heart infusion (BHI) agar for Listeria, and Tryptic Soy Agar (TSA) for E. coli. agar plates had previously been seeded with 0.1 ml of an overnight broth containing 107-108 CFU mL−1 bacterial cultures. The plates were incubated at 37.0 °C for 24 h in an incubation chamber. The diameter of the zone of inhibition was measured with a caliper to the nearest 0.01 mm. The whole zone the area was calculated then subtracted from the film disk area, and this difference in the area was reported as the zone of inhibition. The tests were carried out in triplicate 3
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Fig. 1. Effect of various acid acetic concentration (%) added on the tensile properties of the film, (a) Tensile strength (MPa), (b) Elongation at break (%).
Fig. 2. Effect of various percentages of xylan added on the tensile properties of film, (a) Typical stress-strain curves from films, (b)Tensile strength (MPa), (c) Elongation at Break (%), (d) Young Modulus (GPa).
3.1.2. Effect of xylan percentages The influence of xylan incorporation on the tensile properties of Chitosan-based films has been presented in Fig. 2. The addition of xylan in the considered concentration range significantly affected (P < 0.05) the mechanical properties of the chitosan-based films. The tensile strength of chitosan films increased (P < 0.05) when xylan content increased from 0 wt.% (60.9 MPa) to 25 wt.% (86.2 MPa); the maximum tensile strength values of 94.4 to 101 MPa were achieved on composites films containing 14 to 20 wt.% xylan as shown in Fig. 2 (b). The modulus of the film is represented in function of the amount of xylan added in the composite’s films (Fig. 2 (d)), and clearly show that the rigidity of the composites films increases with the increase of xylan added. The maximum increase is achieved at 20 wt.% xylan added. The
elongation at break shows an increase with the addition of xylan compared to the control chitosan films with maximum increase between 8 and 14 wt.% xylan added as shown in Fig. 2 (c). The difference of elongation at break between 8 and 14 wt.% xylan content was not significantly different, but differently higher than that of control chitosan and chitosan composite with 20 and 25 wt.% xylan. The notable improvement in the tensile properties can be corresponded to the proper dispersion of xylan and carvacol in the chitosan with the aid of Span 80 used as a surfactant. It should be noted that no plasticizer was used in this study in the film making process. The greatest value in elongation at break - 4.5% for the specimen with 8 wt.% xylan - is considerably lower than values reported the previous studies (Schnell et al., 2017; Shen & Kamdem, 4
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2015b) explaining the rigidity of the composites films. However, a significant difference (P < 0.05) in elongation at break was shown for chitosan film and films containing 20 and 25 wt.% xylan, where the lowest value of elongation at break was with chitosan film when no xylan was incorporated. This effect could be explained by the partial replacement of chitosan-chitosan polymer interactions by chitosanxylan interactions in the film network, which may increase the cohesion of the polymer network forces, thus increasing the tensile strength and elongation at break of composite films within the range of conditions of this study. Values of mechanical properties obtained for chitosan and chitosan/ xylan composites films differed from those obtained in other studies (Escalante et al., 2012; Myong, Hwa, Sung-Koo, Curtis, & Milford, 2006; Schnell et al., 2017; Shen & Kamdem, 2015b) and similar to those found by some investigators (Elsabee & Abdou, 2013; Jridi et al., 2014; Martins, Cerqueira, & Vicente, 2012; Thakhiew, Devahastin, & Soponronnarit, 2013). Moreover, the source of chitosan, the acid medium used to disperse the polymer and the experimental conditions (pH, concentration, RH used during equilibration of the film, presence of emulsifiers, type and amount of plasticizers, presence of cross-linking agents) can explain the observed differences (Sánchez-González, Cháfer, Chiralt, & González-Martínez, 2010). The addition of xylan in chitosan film up to 20 wt.% can be used to increase the tensile strength, the elongation at break and the modulus of the resulting film. The increase may be attributed to the interaction between the multiple hydrogen bonds of the hydroxyl groups of xylan and the amine group of chitosan. Above 20% addition, the reduction of properties may result due to saturated chemical interaction and the formation of weaker physical and some repulsive interactions.
thermogravimetric analysis (DTG) of the films in nitrogen environment between 50 and 600 °C at a rate of 10 °C/min are represented in Fig. 3. The films' corresponding degradation temperatures, mass losses (Δw) at different temperature ranges and maximum mass loss rate are presented in Table 2. Previous studies have shown that the thermal degradation of chitosan films occurs a three-step weight loss (Altiok, Altiok, & Tihminlioglu, 2010). Thermogravimetric analysis of xylan reported being a three steps process (Lv, Wu, & Lou, 2010) with potassium xylan softening from 100 to 167 °C followed by a beginning of pyrolysis from 220 to 225 °C and finally pyrolysis at 410 °C. However, four main stages of mass loss were observed in chitosan/ xylan composite films. The first zone occurs between 50 and 110 °C corresponds to the evaporation of water from film and some decomposition of some low molecular weight polymers from Span 80®, carvacrol, xylan and chitosan. The mass loss in this region increases with the adding of xylan in the film formulation when compared to chitosan and remains stable around 8.9 ± 0.2% for all films as presented in Table 2. The mass loss from 3.2 to 4.65% in the second stage of temperature ranging from 110 to 240 °C originated from the mass associated with the softening of xylan, degradation of low molecular weight chitosan, carvacrol, and surfactant. The mass loss at the third stage, which occurred between 220 and 410 °C, was attributed to the initial pyrolysis of xylan and chitosan (Lin, Renneckar, & Hindman, 2007). The fourth zone from 410 to 650 °C is due to the degradation/depolymerization/decomposition of high molecular weight acetylated and deacetylated chitosan and aromatic xylan residues (Tongnuanchan, Benjakul, & Prodpran, 2013; Yang, Yan, Chen, Lee, & Zheng, 2007). The combined mass loss or dry mass loss from 110 to 650 °C in (Table 2) shows a decrease of mass loss with an increase of xylan in the composite. This resulted in more mass residue at the end of the thermal test. The effect of xylan on the initial pyrolysis of composite films can be seen in Fig. 3(c) and (d) as well. As shown in Fig. 3(d) the films containing xylan start to degrade and complete this process at a lower
3.2. Thermal properties of the films Thermogravimetric
analysis
(TGA)
and
derivative
Fig. 3. TGA and DTG curve of chitosan and chitosan/xylan composites films. 5
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Table 2 TGA Weight Loss of Chitosan films containing various amounts of Xylan. Sample ID
Chitosan Xylan 8% Xylan 14% Xylan 20% Xylan 25%
mass loss (%) in each temperature range Stage 1: 50-110 °C
Stage 2: 110-220 °C
Stage 3: 220-410 °C
Stage 4: 410-650 °C
Combined mass loss 110-650 °C
7.96 8.78 9.00 8.99 9.02
3.89 3.45 3.20 4.77 4.65
49.40 50.32 48.99 46.23 45.51
9.45 9.65 9.57 8.72 8.03
62.75 63.42 61.76 60.72 58.19
Mass residue at 650 °C
Degradation temperature at peak (Tmax) (ºC)
Mass loss rate at Tmax (%/ºC)
26.13 24.64 24.81 27.18 28.26
290.72 279.64 275.26 276.82 271.79
0.752 0.759 0.721 0.650 0.607
temperature when compared to the pure chitosan. It is also clear from Fig. 3(d) and Table 2 that the peak positions, which indicate the maximum rate of degradation, shifted to the lower temperature from 290.7 to 271.8 °C by addition of xylan. It should be noted that the sample comprising 20 wt.% xylan showed a peak position at a higher temperature when compared to the 14 and 25 wt.% xylan. This might be due to a better interaction between chitosan and xylan molecules to form more stable moieties. Moreover, the addition of xylan reduced the rate of degradation indicating the composite films degrade at a lower rate with the addition of xylan as shown in Table 2.
Table 3 Antimicrobial activity of films incorporated with different xylan amount. Bacterial type
Sample ID
Inhibition area (mm2)
Antimicrobial Index (%)
L. innocua
Chitosan Xylan 8% Xylan 4% Xylan 20% Xylan 25% Tetracycline Chitosan Xylan 8% Xylan 4% Xylan 20% Xylan 25% Tetracycline
83.83 ± 15.61 93.22 ± 14.38 95.88 ± 10.21 99.92 ± 8.55 99.73 ± 6.72 574.24 ± 19.09 69.85 ± 10.23 65.71 ± 11.66 71.11 ± 10.98 71.20 ± 12.13 73.07 ± 7.95 451.13 ± 17.49
14.60 ± 2.72 16.23 ± 2.50 16.70 ± 1.78 17.40 ± 1.49 17.37 ± 1.17 100 15.48 ± 2.27 14.57 ± 2.59 15.76 ± 2.44 15.78 ± 2.69 16.20 ± 1.76 100
E. coli
3.3. Antibacterial activity Fig. 4 illustrates petri dishes circular disks of chitosan films incorporated with different contents of xylan and carvacrol showing the inhibitory zone against two types of bacteria. the inhibition area in mm2 and the antimicrobial index in a percentage of the films after exposure to the bacterium is also presented in Table 3. It is clear that Tetracycline was very effective with an antimicrobial index of 100% in controlling the growth the bacteria confirming and validating the robustness of the bacteria growth in this study. Besides, chitosan composite films containing 0–25 wt.% xylan and carvacrol showed no significant inhibition to the two tested bacteria. In overall, the antimicrobial activity of the chitosan containing carvacrol and xylan film did not improve significantly (p < 0.05).
mechanical performance was achieved and the thermal behavior and antibacterial activities were also analyzed. To fabricate more ecofriendly materials 50% less acetic acid was used to dissolve chitosan compared to other studies, where only 0.5%v/v acidic acid has been shown to be a sufficient concentration to achieve the maximum toughness. Xylan and carvacrol as ecofriendly materials were added to the chitosan. Adding xylan significantly increased the elongation at break of the composite films and samples with 20 to 25 wt.% xylan exhibited higher tensile strength and young modulus. The results from thermal analysis, which gave useful information for manufacturing processes such as melt compounding, revealed that thermal degradation of chitosan/xylan composite film occurs in four main stages including; I) evaporation of moisture together with low molecular weight polymers, ii) degradation of low molecular weight chitosan, carvacrol, and surfactant, iii) the initial pyrolysis of xylan and chitosan and iv) decomposition of high molecular weight acetylated and deacetylated chitosan and aromatic xylan residues. It also showed that the addition of xylan to chitosan shifts the peak of degradation to a lower
4. Conclusions A biodegradable flexible composite film from renewable resources was developed in this study for food packaging applications. Uniform and homogeneous chitosan-based films containing 0, 8, 14, 20, and 25 wt.% xylan and carvacrol were laboratory hand made. The feasibility of manufacturing was explored and an improvement in the
Fig. 4. Petri dishes circular disks of chitosan films incorporated with different contents of xylan and carvacrol showing the inhibitory zone against two types of bacteria. 6
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temperature, with a lower rate of degradation. Antibacterial activity analysis showed that the addition of carvacrol and xylan weren’t effective; however, tetracycline was very effective in controlling the growth of the bacteria.
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References 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. https://doi.org/10.1007/s10856-010-4065-x. ASTM International (2018). ASTM D882-18 Standard Test Method for Tensile Properties of Thin Plastic Sheeting. West Conshohocken, PA: ASTM Internationalhttps://doi.org/10. 1520/D0882-18. Elsabee, M. Z. E., & Abdou, E. S. (2013). Chitosan based edible films and coatings: A review. Materials Science and Engineering C, 33, 1819–1841. https://doi.org/10.1016/ j.msec.2013.01.010. Escalante, A., Goncalves, A., Bodin, A., Stepan, A., Sandstrom, C., Toriz, G., et al. (2012). Flexible oxygen barrier films from spruce xylan. Carbohydrate Polymers, 87(4), 2381–2387. https://doi.org/10.1016/j.carbpol.2011.11.003. Gordobi, O., Egues, I., Urruzola, I., & labidi, J. (2014). Xylan-cellulose films: Improvement of hydrophobicity, thermal and mechanical properties. Carbohydrate Polymers, 112(4), 56–62. Jamal, S., Zanjani, M., Oğuz, Oğuzhan, Okan, B. S., Yildiz, M., & Menceloğlu, Y. Z. (2019). Polymer composites containing functionalized Nanoparticles and the environment (chapter 14). In K. Pielichowski, & T. M. Majka (Eds.). Micro and nano technologies, polymer composites with functionalized nanoparticles (pp. 437–466). Elsevier ISBN 9780128140642. Jridi, M., Hajji, S., Ayed, H. B., Lassoued, I., Mbarek, A., Kammoun, M., et al. (2014). Physical, structural, antioxidant and antimicrobial properties of gelatin–chitosan composite edible films. International Journal of Biological Macromolecules, 67, 373–379. https://doi.org/10.1016/j.ijbiomac.2014.03.054. Half-Cooked Research Reports (2019). Global plastic packaging market research report forecast to 2022ID: MRFR/PNT/1156-HCRR. Hannah, R. (2018). FAQs on Plastics. Our world in data. https://ourworldindata.org/faqon-plastics. Leceta, I., Guerrero, P., & de la Caba, K. (2013). Functional properties of chitosan-based films. Carbohydrate Polymers, 93(1), 339–346. https://doi.org/10.1016/j.carbpol. 2012.04.031. Lin, Z., Renneckar, S., & Hindman, D. P. (2007). Nanocomposite-based lignocellulosic fibers 1. Thermal stability of modified fibers with clay-polyelectrolyte multilayers. Cellulose, 15(2), 333. https://doi.org/10.1007/s10570-007-9188-y. Lv, G.-J., Wu, S.-B., & Lou, R. (2010). Kinetic study for the thermal decomposition of hemicellulose isolated from corn stalk, Vol. 5https://doi.org/10.15376/biores.5.2.12811291. Martins, J. T., Cerqueira, M. A., & Vicente, A. A. (2012). Influence of α-tocopherol on physicochemical properties of chitosan-based films. Food Hydrocolloids, 27(1), 220–227. https://doi.org/10.1016/j.foodhyd.2011.06.011. Myong, K. K., Hwa, S. J., Sung-Koo, K., Curtis, L. W., & Milford, A. H. (2006). Properties of chitosan films as a function of pH and solvent type. Journal of Food Science, 71(3), E119–E124. https://doi.org/10.1111/j.1365-2621.2006.tb15624.x. Nunes, W. R., Martin, J. R., & Johnson, J. F. (1982). Influence of molecular weight and molecular weight distribution on mechanical properties. Polymer Engineering and
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Development of biodegradable composite chitosan-based films incorporated with xylan and carvacrol for food packaging application
T
Donatien Pascal Kamdema, , Zhu Shena, Omid Nabinejada, Zuju Shub ⁎
a b
School of Packaging, College of Agriculture and Natural Resources, Michigan State University, MI 48824, East Lansing, USA Division of Packaging Engineering, School of Light Textile Engineering and Art, Anhui Agricultural University, Hefei 230036, China
ARTICLE INFO
ABSTRACT
Keywords: Biocomposites Xylan Chitosan Mechanical properties Thermogravimetric analysis Packaging
This paper presents an experimental study on the development of uniform and homogeneous composite chitosan based flexible films containing various amount of xylan from 0 to 25 wt percent and carvacrol. The influence of the addition of xylan on the composite films was evaluated by measuring the tensile strength, moduli and elongation at break. The tensile strength and moduli of composite films containing 20 wt. % increase considerably by 73.9% and 66.8%, respectively. The elongation at break also increases considerably with the addition of xylan, the maximum value of 4.5% was obtained with film containing 8 wt.% xylan. Thermogravimetric analysis (TGA) indicates that the addition of xylan reduces the rate of degradation and shifts the thermal degradation peak from 290.7 to 276.8 °C for the film containing 20 wt.% xylan. Investigation on the antibacterial activity showed that Tetracycline reference control is effective in controlling the growth the bacteria while the incorporation of carvacrol in chitosan xylan composite is not effective.
1. Introduction Plastics derived from fossils fuels have been intensely used for consumers’ products packaging because of their low weight and affordable costs (Tharanathan, 2003). The global plastic packaging market size is projected to reach USD 400 billion by 2022 at a compound annual growth rate (CAGR) of 5% between 2016 and 2022 (HalfCooked Research Reports, 2019). However, the unpredictable price variation of petroleum fossil fuel promotes the variation of the cost of synthetic plastics making the plastic market cost uncertain, which is a non-desirable parameter for packaging manufacturers. The consumers’ awareness of the non-biodegradability, non-compostable packaging and some others negative impact of synthetic plastic packaging at the end of their service life have driven synthetic plastic manufacturers of the global plastic packaging market to find alternative raw materials. It has been reported that the cumulative production of plastics over the period of 1950 to 2015 reached 7.8 billion metric tons - more than one tone of plastic for every person alive today (Hannah, 2018). The majority of them was petroleum-based plastics with low to negligible biodegradability and disposed off yearly into landfills with generation of undesirable byproducts including methane gas. Moreover, greenhouse gas (GHG) and particulates are also released into the surrounding environment during the extraction, transportation, and manufacturing of synthetic plastic from petroleum fossils oils (Robertson, 2008). ⁎
In 2015, Environmental Protection Agency (EPA) reported that 30% of the municipal wastes -mounting to 78 million metric tons were mainly plastic-based packaging materials (Robertson, 2008) with considerable negative impacts on the environment, animal and human life such as irregular climate patterns. Several strategies and means have been proposed to mitigate these changes to sustain the planet. One of the strategies include the uses of biobased available materials or residues with low carbon fingerprint, requiring low processing or manufacturing energy and low emission of greenhouse gas which is the case of several plants and animals residues. Cellulose, hemicellulose, lignin, and starch from plant cell walls are good candidate raw materials from biomass currently used and underdevelopment to make packaging films with some limited properties. Desired packaging material should possess mechanical, optical, thermal properties to offer adequate protection against exterior factors such as vibration, tension, compressions during the logistics. Packaging should also prevent the transfer of unwanted substances such as microbial contamination, moisture, and oxygen to protect the contents (Jamal et al., 2019). They also should be recyclable; renewable, and release low to negligible GHG in the environment (Robertson, 2008). Packaging materials from biological sources including forest, agricultural, plants, animal and seafood residues are known for their biodegradability (Khan et al.2016). They are renewable, available, sometimes edible and relatively low cost and capable to generate sustainable
Corresponding author. E-mail addresses: [email protected] (D.P. Kamdem), [email protected] (Z. Shu).
https://doi.org/10.1016/j.fpsl.2019.100344 Received 17 June 2018; Received in revised form 20 May 2019; Accepted 29 May 2019 Available online 04 June 2019 2214-2894/ © 2019 Published by Elsevier Ltd.
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additional income to fishing or agriculture or forest industries. They are considered residues of another and are considered attractive alternatives to replace fossil fuels plastics packaging materials in several applications. Several researches have focused and reported on the possibility of using chitosan as raw materials for flexible films with properties compared to that of existing synthetic plastic. Chitosan is a cationic natural polymer generated from the de-acetylation of chitin, the major component of the shells of crustaceans (Shen & Kamdem, 2015b). Chitosan is a linear binary heteropolysaccharide composed of β-1,4-linked glucosamine and N-acetylglucosamine, and has been proven to be non-toxic, biodegradable and biocompatible polymer with potential antimicrobial properties (Ojagh, Rezaei, Razavi, & Hosseini, 2010). Chitosan made film is reported to be a good candidate to produce films with permeability to gases (CO2 and O2) comparable to that of commercial compostable poly lactic acid values of 1.99 × 10−17 and 0.49 × 10−17 kg m/m2 s Pa, respectively. Acceptable flexible packaging should also exhibit a tensile stress value of 75 MPa, a tensile modulus and elongation at break close of 2.5 GPa and 75% minimum to be compared to existing commercial materials (Leceta, Guerrero, & de la Caba, 2013; Selke & Culter, 2016). However, development and utilization of chitosan are currently limited because of their low stretchability of 7 to 10% (Shen & Kamdem, 2015a) and their undesirable water relationship in some applications due to the presences of numerous hydroxyl groups. The use of chitosan to produce flexible packaging is limited because of their relatively low tensile strength ranging from 60 to 75 MPa, high water absorption and high oxygen permeability (Shen & Kamdem, 2015b). High water permeability packaging materials is a challenge to protect some fresh and dried food. Water may promote the growth of biodegradable microorganisms such as mold and mildews. To improve the shelf life of the contents, the addition of antimicrobial in the chitosan film during the manufacture may help control the growth of these pathogens. The development of commercially acceptable and performant chitosan-based films will require the increase of the elongation and the reduction of their water vapor retention value to be close to that of flexible films made of synthetic plastic. Several methods have been proposed to improve the barrier and mechanical properties of chitosanbased films including the replacement or substitution of some hydroxyl groups through grafting and/or addition of hydrophobic moieties including fatty acids (Perdones, Vargas, Atarés, & Chiralt, 2014; RuizNavajas, Viuda-Martos, Sendra, Perez-Alvarez, & Fernández-López, 2013; Shen & Kamdem, 2015b). Hemicellulose based films with a tensile strength of up to 100 MPa (Shen & Kamdem, 2015a) much higher than that of some synthetic plastic such as high density polyethylene with 30 MPa tensile strength. It is worth mentioning that hemicellulose is the second most abundant polymer that can be obtained from plants and animal tissues; 15 to 25% of wood is hemicellulose. Natural essential oils such as tung oil, cedarwood oils have shown some potential antimicrobial when included in chitosan film matrix. However, the addition of a hemicellulose such as xylan, which known to have good tensile strength and low oxygen permeability, may improve the properties of composites film xylan (Gordobi, Egues, Urruzola, & labidi, 2014). Hemicellulose based films with a tensile strength of up to 100 MPa (Shen & Kamdem, 2015a) much higher than that of some synthetic plastic such as high density polyethylene with 30 MPa tensile strength. The objective of this study is, therefore, to evaluate the feasibility of manufacturing a composite flexible film for packaging using chitosan as the base polymer matrix, carvacrol to control microbial protection and various amounts of wood xylan from 0 to 25 wt% to improve mechanical properties. The hypothesis of the development of this film formulation with additive proposed will result in the increase of some mechanical and biological properties with the addition of xylan and carvacrol due to the high reactivity of the amine group of chitosan and
the hydroxyl groups from xylan and carvacrol. In addition, the thermal degradation of composite films, which is an important parameter during the manufacturing process such as melt compounding, will also be investigated to figure out the effect of these additions on the thermal stability of compounds. As a consequence of high water-permeability, chitosan film may be susceptible to some bacteria. Therefore, antibacterial activities will also be studied on the films to investigate the usefulness of these composite films for food packaging applications. 2. Materials and methods 2.1. Materials Chitosan from shrimp shells with a deacetylation degree of 75, glacial acetic acid 99%, Span 80®, high purity (98%) carvacrol, and xylan were purchased from Sigma Chemical Co. St. Louis, MO and used without further purification. In our previous study, the molecular weight distribution of chitosan used in this study was determined by gel permeation chromatography (GPC, Waters Model 500 HPLC pump, Milford, USA) with a Millennium software program in water. The number-average (Mn) and weight–average (Mw) molecular weights were 138 and 199 kDa, respectively (Shen & Kamdem, 2015a). Xylan was a light brown solid fine powder extracted from beechwood 95% pure. Carvacrol and xylan were stored in dark container in the laboratory kept at 23 °C. The bacterial strains used in this study were Listeria innocua ATCC33090 (L. innocua), and Escherichia coli ATCC25922 (E. coli). These bacteria were kept at 4 °C. Subcultures of bacteria were carried out every 7 days to maintain bacterial viability. Overnight cultures of these bacteria were grown in Brain-heart infusion (BHI) broth at 37 °C in an incubation chamber from Sheldon Manufacturing Inc, Cornelius, OR. The bacterial population in all the inoculated media was estimated at more than 1 × 107 CFU/ml after 24 h incubation. 2.2. Preparation of the film-forming dispersions Aqueous solution of chitosan was prepared by dissolving 1.5 g of chitosan powder in 148.5 ml of aqueous acetic acid solution representing a 0.5%v/v to obtain a 1%w/v chitosan solution, using a magnetic stirring plate following the method previously published (Shen & Kamdem, 2015b). The chitosan solution was stirred overnight at room temperature to achieve complete dissolution and dispersion. Xylan powder was dissolved in hot water at a solid : liquid ratio of 1:50 during 12 h using magnetic stirring. The solution of xylan in water was filtered to remove the undissolved particulates before mixing with chitosan aqueous solution. Appropriate amounts of xylan solutions were added to the chitosan solution to reach a final concentration of 10, 20, 30, and 40%w/w xylan on the basis of neat chitosan weight. Then 10% w/w of carvacrol, 1%w/w of Span 80® at a level of neat chitosan weight was added to the mixture and stirred for 30 min to achieve a stable well disperse emulsion. Table 1 shows the sample compositions in this study. Finally, emulsions were homogenized with an Ultra-Turrax (IKA, Canada) at 2500 rpm for 4 min. After cooling the resulting mixture at room temperature, it was degassed under vacuum for 5 min in order to Table 1 Sample compositions.
2
Sample ID
Chitosan/ Xylan (%)
Chitosan (g)
Xylan (g)
Carvacrol (g)
Span 80® (g)
Chitosan Xylan 8% Xylan 14% Xylan 20% Xylan 25%
0 10 20 30 40
82.5 76.1 70.8 65.8 61.8
0.0 7.7 14.0 20.0 24.8
8.3 7.6 7.1 6.6 6.2
1.0 1.0 1.0 1.0 1.0
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remove all bubbles. A composite film without xylan was also produced and considered as control. The emulsions were then spread over polystyrene petri dishes (65 mm diameter), which were placed on a leveled surface and allowed to dry for approximately 48 h at 30% RH and 23 °C. Uniform film thicknesses were achieved by casting the same amount of film-forming solution onto each plate. After drying, the films were peeled off the casting surface and maintained at 23 °C and 50% relative humidity (produced with saturated Ca(NO3)2 solution) in a conditioning desiccator until further evaluation.
for each formulation. The agar dilution test was used to determine the density of the bacteria in broth. A serial ten-fold dilutions of broth from 1 to 1 × 10−9 g/ml were made by adding 1 ml of fresh broth into 9 ml of sterile 0.9% sodium chloride solution. After shaking and completely mixing followed by serial dilutions, 1 ml of each solution was sub-cultured on agar plates and dispersed. The settled plates were sealed and placed in an incubator for 24 h. The number of colonies forming units (CFU) of bacteria that appear on the countable agar plate (between 30 and 300) was estimated. For the agar diffusion methods, the films were cut into 10 mm diameter disks with a scissor. Film cuts were placed on Brainheart infusion (BHI) agar for L. innocua and Tryptic Soy Agar (TSA) for E. coli. Films disks specimens were then placed on the surface of inoculated agar petri dish plate, stored in an incubation chamber at 37 °C for 24 h. The area of the inhibition zone defined as zone without apparent visual growth of bacteria was measured with a caliper to the nearest 0.01 mm (Aa). The whole zone area that was pre-inoculated with bacteria was measured and used as the potential surface of bacteria growth and corrected by subtracting the film surface (Ab). The tests were carried out in triplicate for each type of film. Tetracycline was used as a positive control reference well known to inhibit bacteria growth and used to establish the antimicrobial growth. The antimicrobial index was calculated as the percentage of the value of inhibited surface measured with a caliper by the total potential surface growth of inoculated petri dish. A 100% antimicrobial index corresponds to a complete surface inhibition of microbial growth on the petri dish surface while a 0% surface inhibition index is a full microbial growth on the surface of petri dish or 0 cm2 surface inhibition. The antimicrobial index growth for bacteria used in this work was computed by using Eq. (1):
2.3. Film characterization 2.3.1. Film thickness Film thickness was determined using a hand-held digital micrometer (Mitutoyo No. 293-766, Tokyo, Japan) with a precision of 0.001 mm. Measurements were carried out on at least five random locations on each film, and the mean thickness value was calculated and used to calculate the mechanical properties of the films. 2.3.2. Tensile properties of films Standard method D882-12 (ASTM, 2018) was used to measure the tensile properties of films. Films were cut into strips with a test dimension of 50 mm long and 6.35 mm wide. The composite films were determined at 23 and 50%RH with an Instron 5565 Universal Testing Machine (Instron, Canton, MA). The initial gauge length was set to 25 mm and films were stretched using a crosshead speed of 1 mm/min. A microcomputer was used to record the stress-strain curves. Tensile strength and elongation at break were calculated. Four replicates of each test sample were run to achieve dependable data. The Young’s modulus was calculated using Hooke’s law which is the ratio of the stress to the strain in tensile testing and the modulus of elasticity as the initial linear slope of the curves of stress versus strain as related to the equations below:
Antimicrobial index (%) =
Surface inhibited × 100% Total potential surface
(1)
The zero or minimum surface inhibition of bacteria was obtained by using film with no carvacrol content while the maximum inhibition surface was estimated using films containing 2% tetracycline as reference maximum antibacterial (Shen & Kamdem, 2015a).
Young's Modulus (M) = (Stress (MPa) / (Elongation,%) Where; the Young modulus is expressed in force per unit area, the stress in force per unit area and the elongation in percentage of dimension change from the initial dimension. The greater the value of stress or the modulus results in the greater the resistance to deformation or the rigidity of a material. Young modulus is a good indicator of the rigidity of a material while strain or elongation is more related to flexibility and searchability.
3. Results and discussion 3.1. Physical properties of the films 3.1.1. Effect of acetic acid concentration It has been stated that acetic acid used in this study to dissolve chitosan. In order to study the effect of various concentration of acetic acid on the tensile strength and elongation at break is presented in Fig. 1. It is clear from Fig. 1 that the specimen with 0.51% acid acetic exhibits the optimum values for the tensile strength (166.63 MPa), elongation at break (6.72%) and young modulus (6.26 GPa). Since the strength times strain corresponds to the toughness, it can be concluded that the sample with 0.51% acetic acid presents the maximum toughness compared to the other concentration. Therefore, acetic acid with 0.51% concentration has been fixed for the next steps in this study. Chitosan has been previously reported (Xia, Wu, & Chen, 2013) to be solve in 1% (or even more (Shaojie, Xuefeng, Deyong, Yiping, & Kangde, 2004)) acidic acid. The finding from this study indicates that 0.5% acidic acid is appropriate concentration to dissolve chitosan and that chitosan and xylan are miscible in slightly acidic solution to form flexible films. This is important as using less acetic acid results in more ecofriendly materials with better mechanical properties for development of a flexible film based on chitosan. The use of 0.55% at higher acetic acid to dissolve Chitosan may correspond to hydrolysis and depolymerization of chitosan with reduction of strength properties in agreement with literature on the impact of depolymerization on properties of polymers (Nunes, Martin, & Johnson, 1982).
2.3.3. Thermogravimetric analysis Thermogravimetric analysis (TGA) was performed to evaluate the thermal stabilities of composite films using a TGA 2950 running with the Universal Analysis Software Package V.3.9a (TA Instruments, New Castle, DE, USA). Samples of approximately 5 mg were heated from 50 to 650 °C at 10 °C/min heating rate under a nitrogen flow of 70 mL/min. Weight losses of samples were measured as a function of temperature. All the measurements were conducted in duplicate. 2.3.4. Antimicrobial activity The agar diffusion method was used to qualitatively evaluate the antimicrobial activity of the films according to (Pelissari, Grossmann, Yamashita, & Pineda, 2009), with modifications. The films were cut into 10 mm diameter disks with a scissor. Film cuts were placed on Brain-heart infusion (BHI) agar for Listeria, and Tryptic Soy Agar (TSA) for E. coli. agar plates had previously been seeded with 0.1 ml of an overnight broth containing 107-108 CFU mL−1 bacterial cultures. The plates were incubated at 37.0 °C for 24 h in an incubation chamber. The diameter of the zone of inhibition was measured with a caliper to the nearest 0.01 mm. The whole zone the area was calculated then subtracted from the film disk area, and this difference in the area was reported as the zone of inhibition. The tests were carried out in triplicate 3
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Fig. 1. Effect of various acid acetic concentration (%) added on the tensile properties of the film, (a) Tensile strength (MPa), (b) Elongation at break (%).
Fig. 2. Effect of various percentages of xylan added on the tensile properties of film, (a) Typical stress-strain curves from films, (b)Tensile strength (MPa), (c) Elongation at Break (%), (d) Young Modulus (GPa).
3.1.2. Effect of xylan percentages The influence of xylan incorporation on the tensile properties of Chitosan-based films has been presented in Fig. 2. The addition of xylan in the considered concentration range significantly affected (P < 0.05) the mechanical properties of the chitosan-based films. The tensile strength of chitosan films increased (P < 0.05) when xylan content increased from 0 wt.% (60.9 MPa) to 25 wt.% (86.2 MPa); the maximum tensile strength values of 94.4 to 101 MPa were achieved on composites films containing 14 to 20 wt.% xylan as shown in Fig. 2 (b). The modulus of the film is represented in function of the amount of xylan added in the composite’s films (Fig. 2 (d)), and clearly show that the rigidity of the composites films increases with the increase of xylan added. The maximum increase is achieved at 20 wt.% xylan added. The
elongation at break shows an increase with the addition of xylan compared to the control chitosan films with maximum increase between 8 and 14 wt.% xylan added as shown in Fig. 2 (c). The difference of elongation at break between 8 and 14 wt.% xylan content was not significantly different, but differently higher than that of control chitosan and chitosan composite with 20 and 25 wt.% xylan. The notable improvement in the tensile properties can be corresponded to the proper dispersion of xylan and carvacol in the chitosan with the aid of Span 80 used as a surfactant. It should be noted that no plasticizer was used in this study in the film making process. The greatest value in elongation at break - 4.5% for the specimen with 8 wt.% xylan - is considerably lower than values reported the previous studies (Schnell et al., 2017; Shen & Kamdem, 4
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2015b) explaining the rigidity of the composites films. However, a significant difference (P < 0.05) in elongation at break was shown for chitosan film and films containing 20 and 25 wt.% xylan, where the lowest value of elongation at break was with chitosan film when no xylan was incorporated. This effect could be explained by the partial replacement of chitosan-chitosan polymer interactions by chitosanxylan interactions in the film network, which may increase the cohesion of the polymer network forces, thus increasing the tensile strength and elongation at break of composite films within the range of conditions of this study. Values of mechanical properties obtained for chitosan and chitosan/ xylan composites films differed from those obtained in other studies (Escalante et al., 2012; Myong, Hwa, Sung-Koo, Curtis, & Milford, 2006; Schnell et al., 2017; Shen & Kamdem, 2015b) and similar to those found by some investigators (Elsabee & Abdou, 2013; Jridi et al., 2014; Martins, Cerqueira, & Vicente, 2012; Thakhiew, Devahastin, & Soponronnarit, 2013). Moreover, the source of chitosan, the acid medium used to disperse the polymer and the experimental conditions (pH, concentration, RH used during equilibration of the film, presence of emulsifiers, type and amount of plasticizers, presence of cross-linking agents) can explain the observed differences (Sánchez-González, Cháfer, Chiralt, & González-Martínez, 2010). The addition of xylan in chitosan film up to 20 wt.% can be used to increase the tensile strength, the elongation at break and the modulus of the resulting film. The increase may be attributed to the interaction between the multiple hydrogen bonds of the hydroxyl groups of xylan and the amine group of chitosan. Above 20% addition, the reduction of properties may result due to saturated chemical interaction and the formation of weaker physical and some repulsive interactions.
thermogravimetric analysis (DTG) of the films in nitrogen environment between 50 and 600 °C at a rate of 10 °C/min are represented in Fig. 3. The films' corresponding degradation temperatures, mass losses (Δw) at different temperature ranges and maximum mass loss rate are presented in Table 2. Previous studies have shown that the thermal degradation of chitosan films occurs a three-step weight loss (Altiok, Altiok, & Tihminlioglu, 2010). Thermogravimetric analysis of xylan reported being a three steps process (Lv, Wu, & Lou, 2010) with potassium xylan softening from 100 to 167 °C followed by a beginning of pyrolysis from 220 to 225 °C and finally pyrolysis at 410 °C. However, four main stages of mass loss were observed in chitosan/ xylan composite films. The first zone occurs between 50 and 110 °C corresponds to the evaporation of water from film and some decomposition of some low molecular weight polymers from Span 80®, carvacrol, xylan and chitosan. The mass loss in this region increases with the adding of xylan in the film formulation when compared to chitosan and remains stable around 8.9 ± 0.2% for all films as presented in Table 2. The mass loss from 3.2 to 4.65% in the second stage of temperature ranging from 110 to 240 °C originated from the mass associated with the softening of xylan, degradation of low molecular weight chitosan, carvacrol, and surfactant. The mass loss at the third stage, which occurred between 220 and 410 °C, was attributed to the initial pyrolysis of xylan and chitosan (Lin, Renneckar, & Hindman, 2007). The fourth zone from 410 to 650 °C is due to the degradation/depolymerization/decomposition of high molecular weight acetylated and deacetylated chitosan and aromatic xylan residues (Tongnuanchan, Benjakul, & Prodpran, 2013; Yang, Yan, Chen, Lee, & Zheng, 2007). The combined mass loss or dry mass loss from 110 to 650 °C in (Table 2) shows a decrease of mass loss with an increase of xylan in the composite. This resulted in more mass residue at the end of the thermal test. The effect of xylan on the initial pyrolysis of composite films can be seen in Fig. 3(c) and (d) as well. As shown in Fig. 3(d) the films containing xylan start to degrade and complete this process at a lower
3.2. Thermal properties of the films Thermogravimetric
analysis
(TGA)
and
derivative
Fig. 3. TGA and DTG curve of chitosan and chitosan/xylan composites films. 5
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Table 2 TGA Weight Loss of Chitosan films containing various amounts of Xylan. Sample ID
Chitosan Xylan 8% Xylan 14% Xylan 20% Xylan 25%
mass loss (%) in each temperature range Stage 1: 50-110 °C
Stage 2: 110-220 °C
Stage 3: 220-410 °C
Stage 4: 410-650 °C
Combined mass loss 110-650 °C
7.96 8.78 9.00 8.99 9.02
3.89 3.45 3.20 4.77 4.65
49.40 50.32 48.99 46.23 45.51
9.45 9.65 9.57 8.72 8.03
62.75 63.42 61.76 60.72 58.19
Mass residue at 650 °C
Degradation temperature at peak (Tmax) (ºC)
Mass loss rate at Tmax (%/ºC)
26.13 24.64 24.81 27.18 28.26
290.72 279.64 275.26 276.82 271.79
0.752 0.759 0.721 0.650 0.607
temperature when compared to the pure chitosan. It is also clear from Fig. 3(d) and Table 2 that the peak positions, which indicate the maximum rate of degradation, shifted to the lower temperature from 290.7 to 271.8 °C by addition of xylan. It should be noted that the sample comprising 20 wt.% xylan showed a peak position at a higher temperature when compared to the 14 and 25 wt.% xylan. This might be due to a better interaction between chitosan and xylan molecules to form more stable moieties. Moreover, the addition of xylan reduced the rate of degradation indicating the composite films degrade at a lower rate with the addition of xylan as shown in Table 2.
Table 3 Antimicrobial activity of films incorporated with different xylan amount. Bacterial type
Sample ID
Inhibition area (mm2)
Antimicrobial Index (%)
L. innocua
Chitosan Xylan 8% Xylan 4% Xylan 20% Xylan 25% Tetracycline Chitosan Xylan 8% Xylan 4% Xylan 20% Xylan 25% Tetracycline
83.83 ± 15.61 93.22 ± 14.38 95.88 ± 10.21 99.92 ± 8.55 99.73 ± 6.72 574.24 ± 19.09 69.85 ± 10.23 65.71 ± 11.66 71.11 ± 10.98 71.20 ± 12.13 73.07 ± 7.95 451.13 ± 17.49
14.60 ± 2.72 16.23 ± 2.50 16.70 ± 1.78 17.40 ± 1.49 17.37 ± 1.17 100 15.48 ± 2.27 14.57 ± 2.59 15.76 ± 2.44 15.78 ± 2.69 16.20 ± 1.76 100
E. coli
3.3. Antibacterial activity Fig. 4 illustrates petri dishes circular disks of chitosan films incorporated with different contents of xylan and carvacrol showing the inhibitory zone against two types of bacteria. the inhibition area in mm2 and the antimicrobial index in a percentage of the films after exposure to the bacterium is also presented in Table 3. It is clear that Tetracycline was very effective with an antimicrobial index of 100% in controlling the growth the bacteria confirming and validating the robustness of the bacteria growth in this study. Besides, chitosan composite films containing 0–25 wt.% xylan and carvacrol showed no significant inhibition to the two tested bacteria. In overall, the antimicrobial activity of the chitosan containing carvacrol and xylan film did not improve significantly (p < 0.05).
mechanical performance was achieved and the thermal behavior and antibacterial activities were also analyzed. To fabricate more ecofriendly materials 50% less acetic acid was used to dissolve chitosan compared to other studies, where only 0.5%v/v acidic acid has been shown to be a sufficient concentration to achieve the maximum toughness. Xylan and carvacrol as ecofriendly materials were added to the chitosan. Adding xylan significantly increased the elongation at break of the composite films and samples with 20 to 25 wt.% xylan exhibited higher tensile strength and young modulus. The results from thermal analysis, which gave useful information for manufacturing processes such as melt compounding, revealed that thermal degradation of chitosan/xylan composite film occurs in four main stages including; I) evaporation of moisture together with low molecular weight polymers, ii) degradation of low molecular weight chitosan, carvacrol, and surfactant, iii) the initial pyrolysis of xylan and chitosan and iv) decomposition of high molecular weight acetylated and deacetylated chitosan and aromatic xylan residues. It also showed that the addition of xylan to chitosan shifts the peak of degradation to a lower
4. Conclusions A biodegradable flexible composite film from renewable resources was developed in this study for food packaging applications. Uniform and homogeneous chitosan-based films containing 0, 8, 14, 20, and 25 wt.% xylan and carvacrol were laboratory hand made. The feasibility of manufacturing was explored and an improvement in the
Fig. 4. Petri dishes circular disks of chitosan films incorporated with different contents of xylan and carvacrol showing the inhibitory zone against two types of bacteria. 6
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temperature, with a lower rate of degradation. Antibacterial activity analysis showed that the addition of carvacrol and xylan weren’t effective; however, tetracycline was very effective in controlling the growth of the bacteria.
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