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sodium alginate polyelectrolyte films for food packaging materials
Carbohydrate Polymers 170 (2017) 264–270
Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Antimicrobial agent-free hybrid cationic starch/sodium alginate polyelectrolyte films for food packaging materials Ferhat S¸en a,b , I˙ rem Uzunsoy b , Emre Bas¸türk a , Memet Vezir Kahraman a,∗ a b
Marmara University, Department of Chemistry, 34722 Istanbul, Turkey Bülent Ecevit University, Department of Food Processing, 67900 Zonguldak, Turkey
a r t i c l e
i n f o
Article history: Received 15 March 2017 Received in revised form 25 April 2017 Accepted 25 April 2017 Available online 26 April 2017 Keywords: Antimicrobial food packaging Polyelectrolyte Starch Cationic starch Sodium alginate
a b s t r a c t This study aimed to develop polyelectrolyte structured antimicrobial food packaging materials that do not contain any antimicrobial agents. Cationic starch was synthesized and characterized by FT-IR spectroscopy and 1 H NMR spectroscopy. Its nitrogen content was determined by Kjeldahl method. Polyelectrolyte structured antimicrobial food packaging materials were prepared using starch, cationic starch and sodium alginate. Antimicrobial activity of materials was defined by inhibition zone method (disc diffusion method). Thermal stability of samples was evaluated by TGA and DSC. Hydrophobicity of samples was determined by contact angle measurements. Surface morphology of samples was investigated by SEM. Moreover, gel contents of samples were determined. The obtained results prove that produced food packaging materials have good thermal, antimicrobial and surface properties, and they can be used as food packaging material in many industries. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction There are various forms of antimicrobial packages. First, sachet containing antimicrobial agents are added into packages. This is a good commercial practice of antimicrobial packaging. Second, antimicrobial agents are incorporated directly into polymers. In this process, bioactive agents such as silver and zeolite are preferred as antimicrobial agents (Sung et al., 2013). Third, antimicrobials are coated or adsorbed onto polymer surfaces. For example, surface of the polymer can be coated with quaternary ammonium salts or sorbic acid (Xue, Xiao, & Zhang, 2015; Jipa, Guzun, & Stroescu, 2012). Fourth, antimicrobials are immobilized to polymers by ion or covalent linkages. This type of immobilization requires the presence of functional groups on the antimicrobial. For example, peptides, enzymes, polyamines and organic acids types antimicrobial agents contain functional groups. Lastly, some polymers such as chitosan and poly-l-lysine that are inherently (naturally) possess antimicrobial properties and therefore, can be used for food packaging (Fu, Ji, Fan, & Shen, 2006). Polyelectrolytes are polymers like polycations and polyanions, whose repeating units bear an electrolyte group. These groups dissociate in aqueous solutions, making the polymers
∗ Corresponding author. E-mail address: [email protected] (M.V. Kahraman). http://dx.doi.org/10.1016/j.carbpol.2017.04.079 0144-8617/© 2017 Elsevier Ltd. All rights reserved.
charged. Some studies show that polyelectrolytes exhibit antimicrobial properties. For example, Gottenbos, van der Mei, Klatter, Nieuwenhuis, Busscher (2002) prepared positively charged product with silanization of silicone rubber. The obtained products showed antimicrobial effects towards Gram-positive and Gramnegative bacteria (Gottenbos et al., 2002). In another study, Cakmak et al. (2004) prepared cationic polyelectrolytes containing quaternary nitrogen atoms within the main chain via condensation polymerization of epichlorhydrin with benzyl amine. Authors reported that the cationic polyelectrolytes showed antifungal, antibacterial and antiyeast properties (Cakmak, Ulukanli, Tuzcu, Karabuga, & Genctav, 2004). Alginic acid, also called alginate or algin, is an anionic natural polysaccharide. It occurs naturally as the major structural polysaccharide of brown marine algae (Phaeophyceae) and as extracellular mucilage secreted by certain species of bacteria. Therefore, it is renewable, biodegradable, non-toxic, water soluble, abundant and biocompatible (Wang, & Wang, 2010). It is a linear copolymer, constituted of repeating 1,4--d-mannuronopyranosyl and 1,4-␣l-guluronopyranosyl units known as mannuronic and guluronic acid. It tends to be negatively charged and has a wide range of pH values (Harnsilawat, Pongsawatmanit, & McClements, 2006). It is usually present as sodium salt called sodium alginate. Sodium alginates are commonly used as stabilizers, thickeners and gelling agents in several foods, such as soups, deserts, sauces and beverages (Yang, Xie, & He, 2011).
F. S¸en et al. / Carbohydrate Polymers 170 (2017) 264–270
Starch is a semicrystalline polymer. It is a tasteless and odorless powder, mostly obtained from patato and cereals such as wheat, rice and corn. It is constituted of repeating 1,4-␣-d glucopyranosyl units known as amylose and amylopectin. The amounts of amylose and amylopectin units depend on the source that starch is obtained. The properties of starch based materials are closely related to the ratio of these two units. Corn starch contains almost 70% amylopectin and 30% amylose (Liu, Yu, Xie, & Chen, 2006). Starch based materials are of great interest in food packaging sector because of the advantages such as biodegradability, wide availability and the low cost. On the other hand, unfortunately, they have some drawbacks such as poorer mechanical properties and poorer moisture barrier (strong hydrophilic behaviour) than nonbiodegradable polymeric materials used in food packaging industry (Avella et al., 2005). Several starch based materials with antimicrobial properties have been developed (Yoksan, & Chirachanchai, 2010; Corrales, Han, & Tauscher, 2009; Vásconez, Flores, Campos, Alvarado, & Gerschenson, 2009). However, antimicrobial agents are added into these materials. These antimicrobial agents gradually migrate from packaging material to food, which is an unwelcomed situation. In this study, we aimed to develop polyelectrolyte structured antimicrobial food packaging materials that do not contain any antimicrobial agent. Cationic starch was synthesized to obtain cationic groups. Sodium alginate, which is a natural anionic polysaccharide, was used for anionic groups. Polyelectrolyte structured antimicrobial food packaging materials were prepared using starch, cationic starch and sodium alginate. The antimicrobial activity of materials was defined by inhibition zone method (disc diffusion method). Thermal stability, hydrophobicity and gel content of the samples were determined. Surface morphology of the samples was investigated by SEM.
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fine powder. The reaction of preparing cationic starch is shown in Scheme 1a. The nitrogen content of cationic starch was determined by Kjeldahl method. Degree of substitution of cationic starch was calculated according to the following equation. DS =
162xN 1400 − (151.63xN)
where N is nitrogen content determined by Kjeldahl method (%), 162 is the molecular weight of anhydroglucoside unit and 151.63 of GTAC. The degree of substitution of prepared cationic starch was calculated to be 0.35. 2.3. Preparation of polyelectrolyte films Polyelectrolyte structured films were prepared using starch, cationic starch and sodium alginate. Cationic starch was used for cationic groups while sodium alginate was used for anionic groups in polyelectrolyte films. Starch was also used to maintain structural stability in polyelectrolyte films. In addition, glycerol as plasticizer and distilled water as solvent were used. Starch and cationic starch were taken in a beaker and dissolved in 70 mL distilled water. Sodium alginate was dissolved in 30 mL distilled water and added dropwise to the starch mixture. Glycerol was added to the starch − sodium alginate mixture and the reaction mixture was stirred at 100 ◦ C for 10 min. The polyelectrolyte homogenous liquid mixture was poured into a petri dish and dried at 50 ◦ C for 4 days in an oven. Polyelectrolyte films were obtained by removing from the petri dishes. The gel contents of the polyelectrolyte films were found to be between 88% and 95%. The reaction of preparing the polyelectrolyte film is shown in Scheme 1b. The recipes and gel contents of the polyelectrolyte films are shown in Table 1.
2. Experimental
2.4. Measurements and characterization
2.1. Materials
Gel contents of the polyelectrolyte films were determined by Soxhlet extraction for 6 h using acetone. Insoluble gel fraction was dried at 40 ◦ C and the gel content was calculated. Chemical structures of synthesized cationic starch and polyelectrolyte films were examined by Perkin Elmer ATR FT-IR spectrophotometer used in the range of 400–4000 cm−1 . 1 H NMR spectrum of cationic starch was measured on Bruker Advance 500 MHz spectrophotometer. Cationic starch was dissolved in dimethyl sulfoxide before analysis. The antimicrobial activity of produced food packaging materials was defined by inhibition zone method (disc diffusion method). Each bacteria culture was activated by inoculation in Tryptic Soy Broth (TSB), at 37 ◦ C for 24 h. The inoculum (0.1 mL) was spreaded to the surface of Mueller-Hinton (MH) agar petri dishes by spread plate technique, then 6 mm diameter films cut from prepared polyelectrolyte films were placed onto petri dishes. Control samples were prepared under the same conditions. Disc film containing petri dishes and control samples were incubated at 37 ◦ C for 24 h. After incubation, petri dishes were checked for bacterial growth, inhibition zones around the disc films were evaluated qualitatively and quantitatively. Quantitative evaluation was performed according to the inhibiton zone diameter. The zones around the disc films were evaluated as an indicator of inhibiton of bacterial growth. The polyelectrolyte film that produced a large inhibition zone was considered to show a high antimicrobial activity. Thermogravimetric analysis spectra were obtained by Perkin Elmer Pyris-1 model TGA instrument in order to determine thermal behaviour of polyelectrolyte films. The measurement was fulfilled under 2 bar N2 atmosphere and 30–750 ◦ C running range with 10 ◦ C/min heating rate.
Corn starch (unmodified regular corn starch containing approximately 73% amylopectin and 27% amylose), glycidyltrimethylammonium chloride (GTAC), sodium alginate, sodium hydroxide (NaOH) were purchased from Sigma Aldrich. The viscosity of sodium alginate is 5.0–40.0 cps. It is extracted from the alga Macrocystis pyrifera which has an mannuronate/guluronate ratio of 1.6, ˜ & obtained from FT-IR spectroscopic analysis (Gómez-Ordónez Rupérez, 2011). Ethanol was purchased from Merck. All reagents were used without further purification. S. aureus was obtained from microbiology laboratory of Ankara University Dairy Technology Department, and E. coli from Ankara University Food Engineering Department. Bacteria stock cultures were transported to the laboratory in cold storage conditions.
2.2. Synthesis of cationic starch Cationic starch was prepared by attaching positively charged groups onto the H of hydroxyl groups of the starch backbone with NaOH as base catalyst. Firstly, 5 g corn starch, 2.5 g GTAC as cationization reagent, 1.5 mL 1 mol/L NaOH solution and 1.625 mL distilled water were placed in a flask. Then, all the reactants in the flask were well stirred and kept for 5 h at 60 ◦ C in a water bath. The reaction was completed by adding 100 mL of ethanol to the flask and the cationic starch was precipitated. The obtained cationic starch was filtered under vacuum and washed with ethanol twice to remove the unreacted GTAC and NaOH. Product was dried at 50 ◦ C for 6 h in an oven. The obtained white cationic starch was milled to
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Scheme 1. a) Synthesis of cationic starch, b) Preparation of polyelectrolyte films.
Table 1 Recipes, gel contents and contact angle values of the polyelectrolyte films. Sample F1 F2 F3 F4 F5
Starch
Cationic Starch
Sodium Alginate
Glycerol
2 1.5 1.2 1.2 1.0
1 1.5 1.2 0.6 1.0
0.6 1.2 1.0
1.5 1.5 1.5 1.5 1.5
Glass transition temperature of the polyelectrolyte films was determined by DSC analysis using Perkin-Elmer Pyris Diamond differential scanning calorimeter. Samples were heated from 0 to 200 ◦ C, both at a heating rate of 5 ◦ C/min, and cooled at the same rate under nitrogen flow (flow rate 25 mL min−1 ). The surface wettability properties of prepared polyelectrolyte films were investigated by water contact angle () measurements using Kruss (Easy Drop DSA-2) tensiometer. Sessile drop method was used for this analysis. Briefly, 3–5 L of distilled water was pipetted on surface of samples. Image of distilled water drop was captured with a camera and water contact angles of samples were measured by computer software. Morphology of the samples was determined by SEM using Phillips XL 30 ESEM-FEG microscope. Samples were cryo-fractured in liquid nitrogen, covered with a thin gold layer, and examined the fractured surface under SEM.
3.1. FT-IR spectroscopy of cationic starch Fig. 1 shows the FT-IR spectra of starch, GTAC and cationic starch. When the FT-IR spectrum of starch was examined, broad band was observed at 3275 cm−1 , these peaks refer to the hydroxyl group. The band at 2919 cm−1 is the band of the C H stretching vibration. The bands at 1148, 1076 and 996 cm−1 are bands of C O
Contact angle ()
92.4 88.1 93.7 91.2 95.2
72 69 85 88 70
stretching vibrations in the anhydroglucose units of starch (Pal, Mal, & Singh, 2005). When the FT-IR spectrum of GTAC was examined, broad band was observed at 3359 cm−1 ; this peak refers to the hydroxyl group. The band of the C N stretching vibration is observed at 1477 cm−1 . In addition, the band of C H vibration is seen at 3024 cm−1 and the band of epoxy ether vibration is observed at 1264 cm−1 (Wang, & Xie, 2010). When the FT-IR spectrum of cationic starch is examined, it is generally similar to the FT-IR spectrum of unmodified starch. In addition to the characteristic band of starch, band of C N stretching vibration at 1473 cm−1 is seen. But, the band of epoxy ether vibration at 1261 cm−1 in the FT-IR spectrum of GTAC is not observed. These results demonstrate that the desired reaction between starch and GTAC is achieved and cationic starch is successfully synthesized.
3.2. 3. Results and discussion
% Gel content
1H
NMR spectroscopy of cationic starch
Fig. 2 shows 1 H NMR spectrum of cationic starch. It was determined that 1 H-chemical shifts of protons at 3.15–3.64 ppm connecting to the proton at 3.36 ppm to H-4, 3.64 ppm to H-3, 3.31 ppm to H-2 and 3.15 ppm to H-4 (end group). Peaks between 3.38-3.27 ppm and 3.50-3.60 ppm are related to H-6,7,9. The peak associated with methyl protons bound to the nitrogen atom is also observed at 3.20 ppm. The H-1, H-8 and H-5 protons are observed at 5.10 ppm, 4.20 ppm and 4.00 ppm, respectively. Peak of the hydroxyl proton bound to the eighth carbon (OH-8) is seen at
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Fig. 1. FT-IR spectra of starch, GTAC and cationic starch.
Fig. 2.
1
H NMR spectrum of the cationic starch.
5.50 ppm. Decrease in intensity of peaks at 4.58 ppm and 5.40 ppm of the hydroxyl proton bound to the second, third and sixth carbon (OH-2,3,6) and presence of a peak at 3.20 ppm of methyl protons bound to the nitrogen atom proves the formation of the cationic starch (Namazi, Fathi, & Dadkhah, 2011).
3.3. Antimicrobial activity of polyelectrolyte films The antimicrobial activitiy of prepared polyelectrolyte films were tested against both gram positive (S. aureus) and gram negative (E. coli) bacteria. Diameter of inhibition zone of the samples
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Table 2 Antimicrobial activity of polyelectrolyte films against E. coli and S. aureus (inhibition zone diameter in centimeter). Sample
E. coli
S. aureus
F1 F2 F3 F4 F5
1.4 1.8 2.2 1.8 1.7
2.7 2.4 2.6 2.5 2.7
Table 3 Thermal properties of the polyelectrolyte films. Sample
T5% (◦ C)
T50% (◦ C)
Max. weight loss (◦ C)
Char yield (%)
Tg (◦ C)
F1 F2 F3 F4 F5
117 100 147 117 128
303 296 296 274 279
302 316 311 253 278
2.39 4.31 11.43 15.03 15.93
62 63 71 80 –
T5% : temperature at which 5% mass loss is observed. T50% : temperature at which 50% mass loss is observed. Tg : glass transition temperature.
groups in starch. Carboxyl groups of sodium alginate are degraded at lower temperatures than the methylene groups of the starch. This can also be explained by the reduction in cohesive strength and physical intactness between starch and sodium alginate. Char yield of the samples was clearly increased when the cationic starch and sodium alginate content were increased. This increase is related to the nitrogen groups of the cationic starch and sodium groups of sodium alginate (Siddaramaiah Swamy, Ramaraj, & Lee, 2008). DSC technique was used to determine the glass transition temperatures of the prepared polyelectrolyte films. Glass transition temperatures (Tg ) of the samples are given in Table 3. DSC results showed that the addition of cationic starch and sodium alginate increased the Tg values of the samples. It could be due to two reasons: the increase in carboxylate segment concentration by increase of sodium alginate and the loss of mobility of polymer chains due to the decrease in free volume (S¸en, & Kahraman, 2014). 3.5. The surface wettability properties of polyelectrolyte films
are shown in Table 2. It was seen that E. coli and S. aureus grown homogeneously in all regions of petri dishes in control samples. All prepared polyelectrolyte films were found to have inhibitory effects against E. coli and S. aureus. However, the inhibition zone diameter of the samples against S. aureus was greater than the inhibition zone diameter established against E. coli. Therefore, it can be explained that prepared films have more inhibitory effect against gram positive bacteria compared to gram negative bacteria. By increasing the cationic groups in the formulation with cationic starch, it was seen that inhibitory effect of polyelectrolyte films against both E. coli and S. aureus bacteria was increased. On the other hand, it was observed that inhibitory effect of polyelectrolyte system formed by cationic and anionic groups was increased by the addition of anionic groups to the system. Also, the inhibitory effect against bacteria was reduced by decreasing cationic groups and increasing anionic groups in formulations. When all the results were examined, it was observed that the cationic groups were more effective on microorganisms than the anionic groups while polyelectrolyte structures formed by cationic and anionic groups were the best inhibitors.
One of the major disadvantage of starch films is their hydrophilic properties. Several studies have been conducted to improve this property of starch. For example, Kampeerapapp et al. prepared starch/montmorillonite composite films and measured the contact angle of them. They reported that the increase in amount of montmorillonite caused a significant increase in the contact angle values of the films (Kampeerapappun, Aht-ong, Pentrakoon, & Srikulkit, 2007). The surface wettability properties of prepared polyelectrolyte films were investigated by water contact angle measurements. Contact angle values of polyelectrolyte films are given in Table 1. Contact angle of the polyelectrolyte films decreased with an increase in the amount of cationic starch while increased with an increase in the amount of sodium alginate. Contact angle of the F4 sample containing the maximum amount of sodium alginate was 88◦ . This showed that sodium alginate is more hydrophobic than starch and cationic starch. Surface free energy of the materials increases as amount of sodium alginate increases in the formulations. As surface free energies of the materials increase, the contact angle increases and the materials show hydrophobic properties (C¸aykara, Demirci, Ero˘glu, & Güven, 2005). As a result, the hydrophobic properties of polyelectrolyte films were improved by using sodium alginate as the anionic group.
3.4. Thermal properties of polyelectrolyte films
3.6. Morphology of polyelectrolyte films
Thermal oxidative stability of prepared polyelectrolyte films was investigated by TGA technique. Fig. 3 shows the TGA thermograms and data calculated from the thermograms are given in Table 3. These thermograms show that polyelectrolyte films generally undergo thermal degradation at one stage. This indicates that all used materials are compatible with each other. Starch and sodium alginate are hydrophilic and heterocyclic polymers containing −OH groups. Thus, they showed similar thermal degradation. All the samples began to weight lose more than about 100 ◦ C. Degradation between 100 and 200 ◦ C is attributed to the loss of volatile products and it may be due to dehydration. Main degradation of the samples was between 200 and 220 ◦ C. This main weight loss is attributed to the decrosslinking of polymer networks. As a result of the further degradation process, formation of a carbonaceous residue is seen and finally yields Na2 CO3 as char. While There was no significant difference with increasing cationic starch content, main degradation temperature of the samples decreased from 302 ◦ C (F1) to 253 ◦ C (F4) with increasing sodium alginate content. This reduction in main degradation temperature may be attributed to the presence of methylene groups in starch molecules. Because the major structural differences between sodium alginate and starch are carboxyl groups in sodium alginate and methylene
Fig. 4 shows SEM images of the fractured surface of polyelectrolyte films. As seen in Fig. 4 polyelectrolyte films have a smooth, homogeneous and integrated surface. It was seen that there was slight roughness on the surface of the polyelectrolyte films when starch ratio in the formulations decreased. Thus, starch worked as a matrix material in the polyelectrolyte films. Also, results clearly show that all the used materials were compatible with each other. 4. Conclusion This study aimed to develop polyelectrolyte structured antimicrobial food packaging materials that do not contain any antimicrobial agents. Cationic starch was synthesized and characterized by FT-IR and 1 H NMR. The results showed that the desired reaction between starch and GTAC was achieved and the cationic starch was successfully synthesized with a degree of substitution of 0.35. Polyelectrolyte structured antimicrobial food packaging materials were prepared using starch, cationic starch and sodium alginate. Gel contents of prepared polyelectrolyte films were found to be very high. Antimicrobial activity results showed that all prepared polyelectrolyte films have antimicrobial properties. Moreover, the cationic groups were more effective on the
F. S¸en et al. / Carbohydrate Polymers 170 (2017) 264–270
Fig. 3. TGA Spectra of polyelectrolyte films.
Fig. 4. SEM images of polyelectrolyte films.
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controlling microorganisms than the anionic groups while polyelectrolyte structures formed by cationic and anionic groups were the best inhibitors. Prepared polyelectrolyte films showed a good thermal stability, their char yield and glass transition temperature increased with increasing sodium alginate content. Hydrophobic properties of the samples were improved by using sodium alginate. SEM micrographs showed that all used materials were compatible with each other. Finally, the obtained results prove that produced food packaging materials have good thermal, antimicrobial and surface properties, and they can be used as food packaging material in many industries. References C¸aykara, T., Demirci, S., Ero˘glu, M. S., & Güven, O. (2005). Poly(ethylene oxide) and its blends with sodium alginate. Polymer, 46, 10750–10757. S¸en, F., & Kahraman, M. V. (2014). Thermal conductivity and properties of cyanate Ester/nanodiamond composites. Polymers for Advanced Technologies, 25, 1020–1026. Avella, M., Vlieger, J. J. D., Errico, M. E., Fischer, S., Vacca, P., & Volpe, M. G. (2005). Biodegradable starch/clay nanocomposite films for food packaging applications. Food Chemistry, 93, 467–474. Cakmak, I., Ulukanli, Z., Tuzcu, M., Karabuga, S., & Genctav, K. (2004). Synthesis and characterization of novel antimicrobial cationic polyelectrolytes. European Polymer Journal, 40, 2373–2379. Corrales, M., Han, J. H., & Tauscher, B. (2009). Antimicrobial properties of grape seed extracts and their effectiveness after incorporation into pea starch films. International Journal of Food Science and Technology, 44, 425–433. Fu, J., Ji, J., Fan, D., & Shen, J. (2006). Construction of antibacterial multilayer films containing nanosilver via layer-by-layer assembly of heparin and chitosan-silver ions complex. Journal of Biomedical Materials Research Part A, 79A, 665–674. ˜ Gómez-Ordónez, E., & Rupérez, P. (2011). FTIR-ATR spectroscopy as a tool for polysaccharide identification in edible brown and red seaweeds. Food Hydrocolloids, 25(6), 1514–1520. Gottenbos, B., van der Mei, H. C., Klatter, F., Nieuwenhuis, P., & Busscher, H. J. (2002). In vitro and in vivo antimicrobial activity of covalently coupled quaternary ammonium silane coatings on silicone rubber. Biomaterials, 23, 1417–1423.
Harnsilawat, T., Pongsawatmanit, R., & McClements, D. J. (2006). Characterization of b-lactoglobulin-sodium alginate interactions in aqueous solutions: a calorimetry, light scattering, electrophoretic mobility and solubility study. Food Hydrocolloids, 20, 577–585. Jipa, I. M., Guzun, A. S., & Stroescu, M. (2012). Controlled release of sorbic acid from bacterial cellulose based mono and multilayer antimicrobial films. LWT − Food Science and Technology, 47, 400–406. Kampeerapappun, P., Aht-ong, D., Pentrakoon, D., & Srikulkit, K. (2007). Preparation of cassava starch/montmorillonite composite film. Carbohydrate Polymers, 67, 155–163. Liu, H., Yu, L., Xie, F., & Chen, L. (2006). Gelatinization of cornstarch with different amylose/amylopectin content. Carbohydrate Polymers, 65, 357–363. Namazi, H., Fathi, F., & Dadkhah, A. (2011). Hydrophobically modified starch using long-chain fatty acids for preparation of nanosized starch particles. Scientia Iranica C, 18, 439–445. Pal, S., Mal, D., & Singh, R. P. (2005). Cationic starch: an effective flocculating agent. Carbohydrate Polymers, 59, 417–423. Siddaramaiah Swamy, T. M. M., Ramaraj, B., & Lee, J. H. (2008). Sodium alginate and its blends with starch: thermal and morphological properties. Journal of Applied Polymer Science, 109, 4075–4081. Sung, S. Y., Sin, L. T., Tee, T. T., Bee, S. T., Rahmat, A. R., Rahman, W. A. W. A., et al. (2013). Antimicrobial agents for food packaging applications. Trends in Food Science & Technology., 33, 110–123. Vásconez, M. B., Flores, S. K., Campos, C. A., Alvarado, J., & Gerschenson, L. N. (2009). Antimicrobial activity and physical properties of chitosan–tapioca starch based edible films and coatings. Food Research International, 42, 762–769. Wang, W., & Wang, A. (2010). Synthesis and swelling properties of pH-sensitive semi-IPN superabsorbent hydrogels based on sodium alginate-g-poly(sodium acrylate) and polyvinylpyrrolidone. Carbohydrate Polymers, 80, 1028–1036. Wang, Y., & Xie, W. (2010). Synthesis of cationic starch with a high degree of substitution in an ionic liquid. Carbohydrate Polymers, 80, 1172–1177. Xue, Y., Xiao, H., & Zhang, Y. (2015). Antimicrobial polymeric materials with quaternary ammonium and phosphonium salts. International Journal of Molecular Sciences, 16, 3626–3655. Yang, J. S., Xie, Y. J., & He, W. (2011). Research progress on chemical modification of alginate: a review. Carbohydrate Polymers, 84, 33–39. Yoksan, R., & Chirachanchai, S. (2010). Silver nanoparticle-loaded chitosan-starch based films: fabrication and evaluation of tensile, barrier and antimicrobial properties. Materials Science and Engineering C, 30, 891–897.
Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Antimicrobial agent-free hybrid cationic starch/sodium alginate polyelectrolyte films for food packaging materials Ferhat S¸en a,b , I˙ rem Uzunsoy b , Emre Bas¸türk a , Memet Vezir Kahraman a,∗ a b
Marmara University, Department of Chemistry, 34722 Istanbul, Turkey Bülent Ecevit University, Department of Food Processing, 67900 Zonguldak, Turkey
a r t i c l e
i n f o
Article history: Received 15 March 2017 Received in revised form 25 April 2017 Accepted 25 April 2017 Available online 26 April 2017 Keywords: Antimicrobial food packaging Polyelectrolyte Starch Cationic starch Sodium alginate
a b s t r a c t This study aimed to develop polyelectrolyte structured antimicrobial food packaging materials that do not contain any antimicrobial agents. Cationic starch was synthesized and characterized by FT-IR spectroscopy and 1 H NMR spectroscopy. Its nitrogen content was determined by Kjeldahl method. Polyelectrolyte structured antimicrobial food packaging materials were prepared using starch, cationic starch and sodium alginate. Antimicrobial activity of materials was defined by inhibition zone method (disc diffusion method). Thermal stability of samples was evaluated by TGA and DSC. Hydrophobicity of samples was determined by contact angle measurements. Surface morphology of samples was investigated by SEM. Moreover, gel contents of samples were determined. The obtained results prove that produced food packaging materials have good thermal, antimicrobial and surface properties, and they can be used as food packaging material in many industries. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction There are various forms of antimicrobial packages. First, sachet containing antimicrobial agents are added into packages. This is a good commercial practice of antimicrobial packaging. Second, antimicrobial agents are incorporated directly into polymers. In this process, bioactive agents such as silver and zeolite are preferred as antimicrobial agents (Sung et al., 2013). Third, antimicrobials are coated or adsorbed onto polymer surfaces. For example, surface of the polymer can be coated with quaternary ammonium salts or sorbic acid (Xue, Xiao, & Zhang, 2015; Jipa, Guzun, & Stroescu, 2012). Fourth, antimicrobials are immobilized to polymers by ion or covalent linkages. This type of immobilization requires the presence of functional groups on the antimicrobial. For example, peptides, enzymes, polyamines and organic acids types antimicrobial agents contain functional groups. Lastly, some polymers such as chitosan and poly-l-lysine that are inherently (naturally) possess antimicrobial properties and therefore, can be used for food packaging (Fu, Ji, Fan, & Shen, 2006). Polyelectrolytes are polymers like polycations and polyanions, whose repeating units bear an electrolyte group. These groups dissociate in aqueous solutions, making the polymers
∗ Corresponding author. E-mail address: [email protected] (M.V. Kahraman). http://dx.doi.org/10.1016/j.carbpol.2017.04.079 0144-8617/© 2017 Elsevier Ltd. All rights reserved.
charged. Some studies show that polyelectrolytes exhibit antimicrobial properties. For example, Gottenbos, van der Mei, Klatter, Nieuwenhuis, Busscher (2002) prepared positively charged product with silanization of silicone rubber. The obtained products showed antimicrobial effects towards Gram-positive and Gramnegative bacteria (Gottenbos et al., 2002). In another study, Cakmak et al. (2004) prepared cationic polyelectrolytes containing quaternary nitrogen atoms within the main chain via condensation polymerization of epichlorhydrin with benzyl amine. Authors reported that the cationic polyelectrolytes showed antifungal, antibacterial and antiyeast properties (Cakmak, Ulukanli, Tuzcu, Karabuga, & Genctav, 2004). Alginic acid, also called alginate or algin, is an anionic natural polysaccharide. It occurs naturally as the major structural polysaccharide of brown marine algae (Phaeophyceae) and as extracellular mucilage secreted by certain species of bacteria. Therefore, it is renewable, biodegradable, non-toxic, water soluble, abundant and biocompatible (Wang, & Wang, 2010). It is a linear copolymer, constituted of repeating 1,4--d-mannuronopyranosyl and 1,4-␣l-guluronopyranosyl units known as mannuronic and guluronic acid. It tends to be negatively charged and has a wide range of pH values (Harnsilawat, Pongsawatmanit, & McClements, 2006). It is usually present as sodium salt called sodium alginate. Sodium alginates are commonly used as stabilizers, thickeners and gelling agents in several foods, such as soups, deserts, sauces and beverages (Yang, Xie, & He, 2011).
F. S¸en et al. / Carbohydrate Polymers 170 (2017) 264–270
Starch is a semicrystalline polymer. It is a tasteless and odorless powder, mostly obtained from patato and cereals such as wheat, rice and corn. It is constituted of repeating 1,4-␣-d glucopyranosyl units known as amylose and amylopectin. The amounts of amylose and amylopectin units depend on the source that starch is obtained. The properties of starch based materials are closely related to the ratio of these two units. Corn starch contains almost 70% amylopectin and 30% amylose (Liu, Yu, Xie, & Chen, 2006). Starch based materials are of great interest in food packaging sector because of the advantages such as biodegradability, wide availability and the low cost. On the other hand, unfortunately, they have some drawbacks such as poorer mechanical properties and poorer moisture barrier (strong hydrophilic behaviour) than nonbiodegradable polymeric materials used in food packaging industry (Avella et al., 2005). Several starch based materials with antimicrobial properties have been developed (Yoksan, & Chirachanchai, 2010; Corrales, Han, & Tauscher, 2009; Vásconez, Flores, Campos, Alvarado, & Gerschenson, 2009). However, antimicrobial agents are added into these materials. These antimicrobial agents gradually migrate from packaging material to food, which is an unwelcomed situation. In this study, we aimed to develop polyelectrolyte structured antimicrobial food packaging materials that do not contain any antimicrobial agent. Cationic starch was synthesized to obtain cationic groups. Sodium alginate, which is a natural anionic polysaccharide, was used for anionic groups. Polyelectrolyte structured antimicrobial food packaging materials were prepared using starch, cationic starch and sodium alginate. The antimicrobial activity of materials was defined by inhibition zone method (disc diffusion method). Thermal stability, hydrophobicity and gel content of the samples were determined. Surface morphology of the samples was investigated by SEM.
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fine powder. The reaction of preparing cationic starch is shown in Scheme 1a. The nitrogen content of cationic starch was determined by Kjeldahl method. Degree of substitution of cationic starch was calculated according to the following equation. DS =
162xN 1400 − (151.63xN)
where N is nitrogen content determined by Kjeldahl method (%), 162 is the molecular weight of anhydroglucoside unit and 151.63 of GTAC. The degree of substitution of prepared cationic starch was calculated to be 0.35. 2.3. Preparation of polyelectrolyte films Polyelectrolyte structured films were prepared using starch, cationic starch and sodium alginate. Cationic starch was used for cationic groups while sodium alginate was used for anionic groups in polyelectrolyte films. Starch was also used to maintain structural stability in polyelectrolyte films. In addition, glycerol as plasticizer and distilled water as solvent were used. Starch and cationic starch were taken in a beaker and dissolved in 70 mL distilled water. Sodium alginate was dissolved in 30 mL distilled water and added dropwise to the starch mixture. Glycerol was added to the starch − sodium alginate mixture and the reaction mixture was stirred at 100 ◦ C for 10 min. The polyelectrolyte homogenous liquid mixture was poured into a petri dish and dried at 50 ◦ C for 4 days in an oven. Polyelectrolyte films were obtained by removing from the petri dishes. The gel contents of the polyelectrolyte films were found to be between 88% and 95%. The reaction of preparing the polyelectrolyte film is shown in Scheme 1b. The recipes and gel contents of the polyelectrolyte films are shown in Table 1.
2. Experimental
2.4. Measurements and characterization
2.1. Materials
Gel contents of the polyelectrolyte films were determined by Soxhlet extraction for 6 h using acetone. Insoluble gel fraction was dried at 40 ◦ C and the gel content was calculated. Chemical structures of synthesized cationic starch and polyelectrolyte films were examined by Perkin Elmer ATR FT-IR spectrophotometer used in the range of 400–4000 cm−1 . 1 H NMR spectrum of cationic starch was measured on Bruker Advance 500 MHz spectrophotometer. Cationic starch was dissolved in dimethyl sulfoxide before analysis. The antimicrobial activity of produced food packaging materials was defined by inhibition zone method (disc diffusion method). Each bacteria culture was activated by inoculation in Tryptic Soy Broth (TSB), at 37 ◦ C for 24 h. The inoculum (0.1 mL) was spreaded to the surface of Mueller-Hinton (MH) agar petri dishes by spread plate technique, then 6 mm diameter films cut from prepared polyelectrolyte films were placed onto petri dishes. Control samples were prepared under the same conditions. Disc film containing petri dishes and control samples were incubated at 37 ◦ C for 24 h. After incubation, petri dishes were checked for bacterial growth, inhibition zones around the disc films were evaluated qualitatively and quantitatively. Quantitative evaluation was performed according to the inhibiton zone diameter. The zones around the disc films were evaluated as an indicator of inhibiton of bacterial growth. The polyelectrolyte film that produced a large inhibition zone was considered to show a high antimicrobial activity. Thermogravimetric analysis spectra were obtained by Perkin Elmer Pyris-1 model TGA instrument in order to determine thermal behaviour of polyelectrolyte films. The measurement was fulfilled under 2 bar N2 atmosphere and 30–750 ◦ C running range with 10 ◦ C/min heating rate.
Corn starch (unmodified regular corn starch containing approximately 73% amylopectin and 27% amylose), glycidyltrimethylammonium chloride (GTAC), sodium alginate, sodium hydroxide (NaOH) were purchased from Sigma Aldrich. The viscosity of sodium alginate is 5.0–40.0 cps. It is extracted from the alga Macrocystis pyrifera which has an mannuronate/guluronate ratio of 1.6, ˜ & obtained from FT-IR spectroscopic analysis (Gómez-Ordónez Rupérez, 2011). Ethanol was purchased from Merck. All reagents were used without further purification. S. aureus was obtained from microbiology laboratory of Ankara University Dairy Technology Department, and E. coli from Ankara University Food Engineering Department. Bacteria stock cultures were transported to the laboratory in cold storage conditions.
2.2. Synthesis of cationic starch Cationic starch was prepared by attaching positively charged groups onto the H of hydroxyl groups of the starch backbone with NaOH as base catalyst. Firstly, 5 g corn starch, 2.5 g GTAC as cationization reagent, 1.5 mL 1 mol/L NaOH solution and 1.625 mL distilled water were placed in a flask. Then, all the reactants in the flask were well stirred and kept for 5 h at 60 ◦ C in a water bath. The reaction was completed by adding 100 mL of ethanol to the flask and the cationic starch was precipitated. The obtained cationic starch was filtered under vacuum and washed with ethanol twice to remove the unreacted GTAC and NaOH. Product was dried at 50 ◦ C for 6 h in an oven. The obtained white cationic starch was milled to
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Scheme 1. a) Synthesis of cationic starch, b) Preparation of polyelectrolyte films.
Table 1 Recipes, gel contents and contact angle values of the polyelectrolyte films. Sample F1 F2 F3 F4 F5
Starch
Cationic Starch
Sodium Alginate
Glycerol
2 1.5 1.2 1.2 1.0
1 1.5 1.2 0.6 1.0
0.6 1.2 1.0
1.5 1.5 1.5 1.5 1.5
Glass transition temperature of the polyelectrolyte films was determined by DSC analysis using Perkin-Elmer Pyris Diamond differential scanning calorimeter. Samples were heated from 0 to 200 ◦ C, both at a heating rate of 5 ◦ C/min, and cooled at the same rate under nitrogen flow (flow rate 25 mL min−1 ). The surface wettability properties of prepared polyelectrolyte films were investigated by water contact angle () measurements using Kruss (Easy Drop DSA-2) tensiometer. Sessile drop method was used for this analysis. Briefly, 3–5 L of distilled water was pipetted on surface of samples. Image of distilled water drop was captured with a camera and water contact angles of samples were measured by computer software. Morphology of the samples was determined by SEM using Phillips XL 30 ESEM-FEG microscope. Samples were cryo-fractured in liquid nitrogen, covered with a thin gold layer, and examined the fractured surface under SEM.
3.1. FT-IR spectroscopy of cationic starch Fig. 1 shows the FT-IR spectra of starch, GTAC and cationic starch. When the FT-IR spectrum of starch was examined, broad band was observed at 3275 cm−1 , these peaks refer to the hydroxyl group. The band at 2919 cm−1 is the band of the C H stretching vibration. The bands at 1148, 1076 and 996 cm−1 are bands of C O
Contact angle ()
92.4 88.1 93.7 91.2 95.2
72 69 85 88 70
stretching vibrations in the anhydroglucose units of starch (Pal, Mal, & Singh, 2005). When the FT-IR spectrum of GTAC was examined, broad band was observed at 3359 cm−1 ; this peak refers to the hydroxyl group. The band of the C N stretching vibration is observed at 1477 cm−1 . In addition, the band of C H vibration is seen at 3024 cm−1 and the band of epoxy ether vibration is observed at 1264 cm−1 (Wang, & Xie, 2010). When the FT-IR spectrum of cationic starch is examined, it is generally similar to the FT-IR spectrum of unmodified starch. In addition to the characteristic band of starch, band of C N stretching vibration at 1473 cm−1 is seen. But, the band of epoxy ether vibration at 1261 cm−1 in the FT-IR spectrum of GTAC is not observed. These results demonstrate that the desired reaction between starch and GTAC is achieved and cationic starch is successfully synthesized.
3.2. 3. Results and discussion
% Gel content
1H
NMR spectroscopy of cationic starch
Fig. 2 shows 1 H NMR spectrum of cationic starch. It was determined that 1 H-chemical shifts of protons at 3.15–3.64 ppm connecting to the proton at 3.36 ppm to H-4, 3.64 ppm to H-3, 3.31 ppm to H-2 and 3.15 ppm to H-4 (end group). Peaks between 3.38-3.27 ppm and 3.50-3.60 ppm are related to H-6,7,9. The peak associated with methyl protons bound to the nitrogen atom is also observed at 3.20 ppm. The H-1, H-8 and H-5 protons are observed at 5.10 ppm, 4.20 ppm and 4.00 ppm, respectively. Peak of the hydroxyl proton bound to the eighth carbon (OH-8) is seen at
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Fig. 1. FT-IR spectra of starch, GTAC and cationic starch.
Fig. 2.
1
H NMR spectrum of the cationic starch.
5.50 ppm. Decrease in intensity of peaks at 4.58 ppm and 5.40 ppm of the hydroxyl proton bound to the second, third and sixth carbon (OH-2,3,6) and presence of a peak at 3.20 ppm of methyl protons bound to the nitrogen atom proves the formation of the cationic starch (Namazi, Fathi, & Dadkhah, 2011).
3.3. Antimicrobial activity of polyelectrolyte films The antimicrobial activitiy of prepared polyelectrolyte films were tested against both gram positive (S. aureus) and gram negative (E. coli) bacteria. Diameter of inhibition zone of the samples
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Table 2 Antimicrobial activity of polyelectrolyte films against E. coli and S. aureus (inhibition zone diameter in centimeter). Sample
E. coli
S. aureus
F1 F2 F3 F4 F5
1.4 1.8 2.2 1.8 1.7
2.7 2.4 2.6 2.5 2.7
Table 3 Thermal properties of the polyelectrolyte films. Sample
T5% (◦ C)
T50% (◦ C)
Max. weight loss (◦ C)
Char yield (%)
Tg (◦ C)
F1 F2 F3 F4 F5
117 100 147 117 128
303 296 296 274 279
302 316 311 253 278
2.39 4.31 11.43 15.03 15.93
62 63 71 80 –
T5% : temperature at which 5% mass loss is observed. T50% : temperature at which 50% mass loss is observed. Tg : glass transition temperature.
groups in starch. Carboxyl groups of sodium alginate are degraded at lower temperatures than the methylene groups of the starch. This can also be explained by the reduction in cohesive strength and physical intactness between starch and sodium alginate. Char yield of the samples was clearly increased when the cationic starch and sodium alginate content were increased. This increase is related to the nitrogen groups of the cationic starch and sodium groups of sodium alginate (Siddaramaiah Swamy, Ramaraj, & Lee, 2008). DSC technique was used to determine the glass transition temperatures of the prepared polyelectrolyte films. Glass transition temperatures (Tg ) of the samples are given in Table 3. DSC results showed that the addition of cationic starch and sodium alginate increased the Tg values of the samples. It could be due to two reasons: the increase in carboxylate segment concentration by increase of sodium alginate and the loss of mobility of polymer chains due to the decrease in free volume (S¸en, & Kahraman, 2014). 3.5. The surface wettability properties of polyelectrolyte films
are shown in Table 2. It was seen that E. coli and S. aureus grown homogeneously in all regions of petri dishes in control samples. All prepared polyelectrolyte films were found to have inhibitory effects against E. coli and S. aureus. However, the inhibition zone diameter of the samples against S. aureus was greater than the inhibition zone diameter established against E. coli. Therefore, it can be explained that prepared films have more inhibitory effect against gram positive bacteria compared to gram negative bacteria. By increasing the cationic groups in the formulation with cationic starch, it was seen that inhibitory effect of polyelectrolyte films against both E. coli and S. aureus bacteria was increased. On the other hand, it was observed that inhibitory effect of polyelectrolyte system formed by cationic and anionic groups was increased by the addition of anionic groups to the system. Also, the inhibitory effect against bacteria was reduced by decreasing cationic groups and increasing anionic groups in formulations. When all the results were examined, it was observed that the cationic groups were more effective on microorganisms than the anionic groups while polyelectrolyte structures formed by cationic and anionic groups were the best inhibitors.
One of the major disadvantage of starch films is their hydrophilic properties. Several studies have been conducted to improve this property of starch. For example, Kampeerapapp et al. prepared starch/montmorillonite composite films and measured the contact angle of them. They reported that the increase in amount of montmorillonite caused a significant increase in the contact angle values of the films (Kampeerapappun, Aht-ong, Pentrakoon, & Srikulkit, 2007). The surface wettability properties of prepared polyelectrolyte films were investigated by water contact angle measurements. Contact angle values of polyelectrolyte films are given in Table 1. Contact angle of the polyelectrolyte films decreased with an increase in the amount of cationic starch while increased with an increase in the amount of sodium alginate. Contact angle of the F4 sample containing the maximum amount of sodium alginate was 88◦ . This showed that sodium alginate is more hydrophobic than starch and cationic starch. Surface free energy of the materials increases as amount of sodium alginate increases in the formulations. As surface free energies of the materials increase, the contact angle increases and the materials show hydrophobic properties (C¸aykara, Demirci, Ero˘glu, & Güven, 2005). As a result, the hydrophobic properties of polyelectrolyte films were improved by using sodium alginate as the anionic group.
3.4. Thermal properties of polyelectrolyte films
3.6. Morphology of polyelectrolyte films
Thermal oxidative stability of prepared polyelectrolyte films was investigated by TGA technique. Fig. 3 shows the TGA thermograms and data calculated from the thermograms are given in Table 3. These thermograms show that polyelectrolyte films generally undergo thermal degradation at one stage. This indicates that all used materials are compatible with each other. Starch and sodium alginate are hydrophilic and heterocyclic polymers containing −OH groups. Thus, they showed similar thermal degradation. All the samples began to weight lose more than about 100 ◦ C. Degradation between 100 and 200 ◦ C is attributed to the loss of volatile products and it may be due to dehydration. Main degradation of the samples was between 200 and 220 ◦ C. This main weight loss is attributed to the decrosslinking of polymer networks. As a result of the further degradation process, formation of a carbonaceous residue is seen and finally yields Na2 CO3 as char. While There was no significant difference with increasing cationic starch content, main degradation temperature of the samples decreased from 302 ◦ C (F1) to 253 ◦ C (F4) with increasing sodium alginate content. This reduction in main degradation temperature may be attributed to the presence of methylene groups in starch molecules. Because the major structural differences between sodium alginate and starch are carboxyl groups in sodium alginate and methylene
Fig. 4 shows SEM images of the fractured surface of polyelectrolyte films. As seen in Fig. 4 polyelectrolyte films have a smooth, homogeneous and integrated surface. It was seen that there was slight roughness on the surface of the polyelectrolyte films when starch ratio in the formulations decreased. Thus, starch worked as a matrix material in the polyelectrolyte films. Also, results clearly show that all the used materials were compatible with each other. 4. Conclusion This study aimed to develop polyelectrolyte structured antimicrobial food packaging materials that do not contain any antimicrobial agents. Cationic starch was synthesized and characterized by FT-IR and 1 H NMR. The results showed that the desired reaction between starch and GTAC was achieved and the cationic starch was successfully synthesized with a degree of substitution of 0.35. Polyelectrolyte structured antimicrobial food packaging materials were prepared using starch, cationic starch and sodium alginate. Gel contents of prepared polyelectrolyte films were found to be very high. Antimicrobial activity results showed that all prepared polyelectrolyte films have antimicrobial properties. Moreover, the cationic groups were more effective on the
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Fig. 3. TGA Spectra of polyelectrolyte films.
Fig. 4. SEM images of polyelectrolyte films.
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controlling microorganisms than the anionic groups while polyelectrolyte structures formed by cationic and anionic groups were the best inhibitors. Prepared polyelectrolyte films showed a good thermal stability, their char yield and glass transition temperature increased with increasing sodium alginate content. Hydrophobic properties of the samples were improved by using sodium alginate. SEM micrographs showed that all used materials were compatible with each other. Finally, the obtained results prove that produced food packaging materials have good thermal, antimicrobial and surface properties, and they can be used as food packaging material in many industries. References C¸aykara, T., Demirci, S., Ero˘glu, M. S., & Güven, O. (2005). Poly(ethylene oxide) and its blends with sodium alginate. Polymer, 46, 10750–10757. S¸en, F., & Kahraman, M. V. (2014). Thermal conductivity and properties of cyanate Ester/nanodiamond composites. Polymers for Advanced Technologies, 25, 1020–1026. Avella, M., Vlieger, J. J. D., Errico, M. E., Fischer, S., Vacca, P., & Volpe, M. G. (2005). Biodegradable starch/clay nanocomposite films for food packaging applications. Food Chemistry, 93, 467–474. Cakmak, I., Ulukanli, Z., Tuzcu, M., Karabuga, S., & Genctav, K. (2004). Synthesis and characterization of novel antimicrobial cationic polyelectrolytes. European Polymer Journal, 40, 2373–2379. Corrales, M., Han, J. H., & Tauscher, B. (2009). Antimicrobial properties of grape seed extracts and their effectiveness after incorporation into pea starch films. International Journal of Food Science and Technology, 44, 425–433. Fu, J., Ji, J., Fan, D., & Shen, J. (2006). Construction of antibacterial multilayer films containing nanosilver via layer-by-layer assembly of heparin and chitosan-silver ions complex. Journal of Biomedical Materials Research Part A, 79A, 665–674. ˜ Gómez-Ordónez, E., & Rupérez, P. (2011). FTIR-ATR spectroscopy as a tool for polysaccharide identification in edible brown and red seaweeds. Food Hydrocolloids, 25(6), 1514–1520. Gottenbos, B., van der Mei, H. C., Klatter, F., Nieuwenhuis, P., & Busscher, H. J. (2002). In vitro and in vivo antimicrobial activity of covalently coupled quaternary ammonium silane coatings on silicone rubber. Biomaterials, 23, 1417–1423.
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