International Journal of Biological Macromolecules 80 (2015) 596–604
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Performance properties and antibacterial activity of crosslinked films of quaternary ammonium modified starch and poly(vinyl alcohol) Zahra Sekhavat Pour, Pooyan Makvandi, Mousa Ghaemy ∗ Polymer Research Laboratory, Department of Chemistry, University of Mazandaran, Babolsar, Iran
a r t i c l e
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Article history: Received 11 May 2015 Received in revised form 20 June 2015 Accepted 8 July 2015 Available online 15 July 2015 Keywords: Poly(vinyl alcohol) Antibacterial activity Biomaterial Quaternary ammonium starch Chemical crosslinking
a b s t r a c t There has been a growing interest in developing antibacterial polymeric materials. In the present work, novel antibacterial cross-linked blend films were prepared based on polyvinyl alcohol (PVA) and quaternary ammonium starch (ST-GTMAC) using citric acid (CA) as plasticizer and glutaraldehyde (GA) as cross-linker. The ST-GTMAC was successfully synthesized from reaction between water-soluble oxidized starch and glycidyltrimethylammonium chloride (GTMAC). The effect of ST-GTMAC, CA and GA contents on the swelling, solubility, mechanical and thermal properties of the films was investigated. It was found that incorporation of ST-GTMAC reduced UV-transmittance and provided antibacterial properties, increasing GA content increased tensile strength and decreased solubility and swelling degree of the films, while CA acted as plasticizer when its concentration was above 10 wt%. The results showed that ST-GTMAC/PVA/CA/GA film has fair antibacterial activity against Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. These results suggest that the prepared film might be used as potential antibacterial material in medical and packaging applications. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Biodegradable plastics have attracted growing attention because of their potential use in the replacement of traditional petroleum-based synthetic polymers that do not degrade in a land fill and poses a serious environmental threat. Poly(vinyl alcohol) (PVA) is recognized as one of the very few vinyl polymers soluble in water also susceptible of biodegradation in the presence of suitably acclimated microorganisms. However, PVA has poor dimensional stability due to high moisture absorption and relatively high price compared to other commercial polymers. Therefore, increasing attention is devoted to the preparation of environmentally compatible PVA-based materials for a wide range of applications such as wound dressing material, various coatings, and active packaging including controlled delivery systems [1–4]. Among all natural biopolymers, PVA-blended with renewable and abundant polysaccharide such as starch can be most promising because of its easy availability, biodegradability, and lower cost. Starch has been used as a filler to produce reinforced plastics to reduce environmental pollution [5,6]. PVA/starch blend is suitable to be used as
∗ Corresponding author. E-mail address: [email protected] (M. Ghaemy). http://dx.doi.org/10.1016/j.ijbiomac.2015.07.008 0141-8130/© 2015 Elsevier B.V. All rights reserved.
biodegradable packaging and agricultural materials [7]. Their physical properties can be improved by adding plasticizers such as glycerol [7,8], by using chemically modified starch [9,10], by adding crosslinker [3,11], or by preparing biodegradable nanocomposites [12,13]. Antimicrobial packaging has one of the most promising active packaging systems which improve the food safety and shelf-life. There are several ways in which antibacterial properties can be accomplished including direct incorporation of an antimicrobial agent into a polymer, coating or adsorbing an antibacterial agent onto the polymer surface, immobilization of an antibacterial agent in the polymer via ionic or covalent bonding, etc. [1]. The immobilization of quaternary ammonium salt was proved to be an efficient approach to impose antibacterial property on a biomaterial [14,15]. Glycidyltrimethylammonium chloride (GTMAC) is a quaternary ammonium salt that has received much attention as an antibacterial agent [16,17], and its antibacterial activity in chitosan was investigated after preparation of crosslinked blend films with PVA [16]. However, in our knowledge, there has been no study on preparation, characterization and antibacterial activity of crosslinked blend film composed of quaternary ammonium modified starch as a low cost biomaterial and PVA. Therefore, the aim of this work was to study the antimicrobial activity of the crosslinked blend films of PVA with starch chemically bonded with antimicrobial agent,
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glycidyl trimethylammonium chloride (GTMAC). The antimicrobial functionalized starch (ST-GTMAC) was prepared by reacting water soluble oxidized starch with GTMAC, and then ST-GTMAC was blended with PVA, plasticized by citric acid (CA) and chemically crosslinked by glutaraldehyde (GA). The effect of ST-GTMAC, GA and CA contents in the blend films was investigated on the physical properties such as tensile strength, thermal properties, swelling behavior, solubility, and UV–vis protection. Also, antibacterial activity of the films was investigated against Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. 2. Experimental 2.1. Materials Starch, polyvinyl alcohol (PVA, 98% hydrolyzed with a molecular weight average of 49,000), sodium bromate, trimethylamine solution (33 wt% in ethanol), glutaraldehyde (GA) aqueous solution (25 wt%), epichlorohydrin and citric acid (CA) were purchased from Merk (Germany) and used without further purification. 2.2. Oxidation of starch Oxidized starch was synthesized according the procedure described elsewhere [18]. Briefly, 3 g starch, 15 mL distilled water, and 1.05 equivalent NaBrO3 (2.927 g) per glucose unit were mixed in a 500 mL flask. Then, 5.5 mL (1.77 M) H2 SO4 was manually added into the flask within 60 min at room temperature. The reaction was stopped after 24 h by neutralizing the solution with NaOH (1 M) until pH 9. The oxidation product was precipitated by addition of ethanol, and then washed three times with a water-ethanol mixture (30 mL of distilled H2 O and 300 mL of cold ethanol) and dried in a vacuum oven at 30 ◦ C for 24 h. Back titration was used to determine the carboxyl group content of oxidized starch. Starch solution (0.5 g dry oxidized starch in 300 mL distilled water) was adjusted to pH 2.5 with 0.1 M HCl at room temperature, stirred for 15 min and titrated to pH 8.3 with standardized 0.1 M NaOH. Starch was used as blank sample. Carboxyl content of the sample was calculated as follows [18]: Percentage of carboxyl content =
[(Sample-blank) mL × molarity of NaOH × 100 × 0.045] Sample weight in grams
(1)
The content of carboxyl groups of the oxidized starch was about 10.36 ± 0.86% by titration method. 2.3. Preparation of glycidyltrimethylammonium chloride (GTMAC) In a three-necked round bottom flask equipped with a magnetic stir, a condenser, and nitrogen gas inlet tube, 4.0 mmol trimethylamine was added to 12.0 mmol epichlorohydrin in 20 mL ethanol. The solution was then heated to reflux for 24 h. After evaporation of the solvent under reduced pressure, quaternary ammonium salt was purified by recrystallization from acetone. (2,3-epoxypropyl) trimethylammonium chloride (GTMAC) was obtained by raising the pH of quaternary ammonium salt aqueous solution to 8 by using 15% (w/v) NaOH [19]. 2.4. Preparation of (2-hydroxy)propyl-3-trimethylammonium starch chloride (ST-GTMAC) In a three-necked round bottom flask equipped with a magnetic stirer, a condenser, and nitrogen gas inlet tube, 1.5 g oxidized
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starch (ST) was dissolved in distilled water (20 mL) and then 0.5 g GTMAC and catalytic amount of triethylamine solution (25% v/v) were added. The reaction was carried out under nitrogen at 70 ◦ C for 24 h. Then, the solution was poured into acetone/methanol (50/50, v/v) mixture to obtain the precipitate (ST-GTMAC) which was filtered and washed with acetone several times. Finally, ST-GTMAC was extracted with hot methanol in a soxhlet apparatus for 48 h to remove unreacted glycidyltrimethylammonium chloride. 2.5. Preparation of PVA/ST-GTMAC crosslinked film Polymer films were prepared by solution casting method. PVA was dissolved in distilled water (at 5% w/v) in a flask under magnetic stirring at 90 ◦ C for 3 h. ST-GTMAC was added at different mass ratios where part of the PVA was gradually replaced by ST-GTMAC up to 40%. Then citric acid (0–30 wt% based on the weight of polymer blend) as plasticizer was added and the mixture was stirred for 2 h at 40 ◦ C, the temperature was raised to 65 ◦ C and glutaraldehyde (0.25–3 wt% based on the weight of polymer blend) as crosslinking agent was gradually added and the solution was stirred for 15 min. Then the solution was cooled down and poured into the Petri dish to be dried at 35 ◦ C to form the crosslinked PVA/ST-GTMAC blend film. The crosslinked film was extracted with hot methanol in a soxhlet apparatus for 24 h to remove unreacted GA, and dried in a vacuum oven at 60 ◦ C. 2.6. Characterization 2.6.1. FTIR and 1 H NMR Fourier-transform infrared (FT-IR) spectra were recorded on a Bruker Vector 22 FTIR spectrometer in the range of 400–4000 cm−1 . 1 H NMR (400 MHz) spectra were obtained on a Bruker DRX 400 Advance spectrometer at room temperature with D2 O as solvent and tetramethylsilane (TMS) as the internal standard. Chemical shifts are reported in ppm relative to the deuterated solvent resonances. 2.6.2. Swelling degree and solubility of films The percentage of degree of swelling (DS%) and solubility (S%) of the films was measured using the following method. Dried PVA/STGTMAC crosslinked blend films were immersed in distilled water at room temperature. After the equilibrium (24 h), moisture on the surface of the film was removed and the weight of the film was measured. DS of the film was calculated using Eq. (2). DS% =
(We − W0 ) × 100 W0
(2)
where We is the weight of the film at the adsorbing equilibrium, and W0 is the initial dry weight of the film. The swollen film was dried again for 24 h at 30 ◦ C, and its solubility (S) was calculated using Eq. (3). S% =
W0 − Wd × 100 W0
(3)
where W0 represents the initial weight of the specimen and Wd is the weight after drying process. Each experiment was performed in triplicate. 2.6.3. Mechanical properties of films Tensile strength (TS) and elongation at break (El@br) were measured for each crosslinked film by Instron Universal Testing Machine (Model DBBP 500, BONGSHIN). Three rectangular shaped specimens were cut out from each film and each specimen was 12 mm wide with an average thickness of 0.05 mm. The gauge length and grip distance were both 50.0 mm. Crosshead speed was 5 mm min−1 and load cell was 250 kgf.
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Fig. 1. Synthetic approach of quaternary ammonium starch (ST-GTMAC).
2.6.4. Thermal properties The thermal behavior of crosslinked blend films was evaluated by thermal gravimetrical analysis (TGA) (Rheometric Scientific, USA) from room temperature up to 600 ◦ C with the heating rate of 10 ◦ C min−1 . A differential scanning calorimetry (DSC) (DSC1 METTLER TOLEDO, Switzerland) was employed to investigate thermal properties of the crosslinked blend films. The sample (3–10 mg) was placed in an aluminum pan and measurement was carried out under nitrogen flow in the temperature range from 20 to 220 ◦ C at 10 ◦ C min−1 . 2.6.5. Ultraviolet–visible spectrophotometer (UV–vis) UV–vis absorption spectra of the samples (1 × 1 cm2 ) were recorded in the wavelength of 250–700 nm using a Perkin Elmer Instruments (Lambda 35) UV–vis 141 spectrophotometer. The transparency value of the crosslinked films was calculated using Eq. (4) [20]. Transparency = −(log T600 )/x
(4)
where T600 is the fractional transmittance at 600 nm and x is the film thickness (mm). The greater value represents the lower transparence of the film.
2.6.6. Antibacterial activity assay The in vitro antibacterial activities of the compounds were assayed using direct contact test with agar diffusion. The test compounds were prepared as film with a 7 mm in diameter then placed on the surface of inoculated agar plates [21]. The antibacterial activity of the compounds was investigated against four bacterial. Test organisms included E. coli PTCC 1330, P. aeruginosa PTCC 1074, S. aureus ATCC 35923, B. subtilis PTCC 1023. All tested bacteria were maintained in Mueller-Hinton broth (Merck). Before using the cultures, they were standardized with a final cell density of approximately 108 cfu mL−1 . For the direct contact agar diffusion test, the Mueller-Hinton agar plate (Merck) media were used. The agar plates were inoculated from the standardized cultures of the test organisms using a sterile cotton swab then spread as uniformly as possible throughout the entire media. The film was introduced on the upper layer of the seeded agar plate. Mitis-salivaris agar plate was incubated at 37 ◦ C for 72–96 h supplied with 5% CO2 , SABOURAUD Dextrose agar plate and Mueller-Hinton agar plate were incubated at 37 ◦ C for 24–48 h. The antibacterial activities of the compounds were compared with known antibiotic gentamicin (10 g disk−1 ), chloramphenicol (30 g disk−1 ). Positive control plates were streaked with test organisms, but no film was
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used. Antibacterial activity was evaluated by measuring the diameter of inhibition zone (mm) on the surface of plates and the results were reported as mean ± SD after three repeats.
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are assigned to methine proton (b), methylene protons (c), and methylene protons (a), respectively [25], as indicated in Fig. 1S. 3.2. Appearance of PVA/ST-GTMAC crosslinked films
3. Results and discussion 3.1. Synthesis and characterization of ST-GTMAC The synthetic approach for the preparation of ST-GTMAC is illustrated in Fig. 1. Introduction of carboxylic groups (10.36 ± 0.86%) in the oxidized starch structure made starch to dissolve in water completely. In quaternization reaction, the hydroxyl and carboxylic acid groups of the oxidized starch can react with the epoxide ring of glycidyltrimethylammonium chloride (GTMAC) leading to grafting of quaternary ammonium salt moiety onto macromolecular chains of starch to give ST-GTMAC. FT-IR spectra of starch, oxidized starch and ST-GTMAC are shown in Fig. 2. In the spectrum of starch, the absorption bands in the region of 700–1000 cm−1 are due to atomic group involved in the anomeric form [22], in the region of 1000–1200 cm−1 are due to stretching vibration of C O, and in the region of 1200–1500 cm−1 are related to C H bending [23]. The absorption bands in regions of 2800–3000 cm−1 and 3300–3500 cm−1 are assigned to C H and O H stretching, respectively. In the spectrum of oxidized starch, two strong absorption bands are observed at 1613 and 1419 cm−1 due to carboxylate group which is due to oxidation of starch [18,24]. In the FT-IR spectrum of GTMAC, the absorption bands at around 948 cm−1 are the characteristic of epoxide group. Absorption bands in the region of 600–1000 cm−1 are due to stretching vibrations of the NR4 + complexes. The main absorption band of GTMAC is at 1480 cm−1 due to C H symmetric bending of the methyl groups on the quaternary ammonium substituent [17,25]. FT-IR spectrum of ST-GTMAC is similar to that of oxidized starch except for the presence of an absorption band at 1480 cm−1 due to methyl groups on the quaternary ammonium substituent. These results confirm that quaternary ammonium moieties have been chemically attached to the macromolecular chains of starch. In the 1 H NMR spectrum of oxidized starch (Fig. 1S), signals were observed at 5.48 ppm related to the anomeric proton of anhydroglucose unit, other anomeric protons were appeared at 4.8–5.54 ppm, and signals of ring protons were observed in the range of 3.5–4.19 [18]. The characteristic signal of ST-GTMAC was appeared at 3.18 ppm, which is related to the protons of trimethylammonium group (d) [17]. Other signals at 4.22, 3.45 and 2.2 ppm
Fig. 2. FT-IR spectrum of starch, oxidized starch, GTMAC and ST-GTMAC.
Preparation of PVA/ST-GTMAC crosslinked films was carried out by mixing a solution of different weight ratios of PVA, STGTMAC, citric acid (CA) as plasticizer, and glutaraldehyde (GA) as crosslinking agent. Then clear films were prepared by solution casting method. Fig. 3a shows transparency of PVA film, PVA/STGTMAC10%/GA2%/CA25%, and PVA/ST-GTMAC30%/GA2%/CA25% crosslinked films. As can be seen in Fig. 3a, PVA film is colorless and transparent but incorporation of ST-GTMAC up to 30 wt% has changed the color of the film slightly to yellowish. Both PVA and ST-GTMAC macromolecular chains are involved in the crosslink reactions with GA, as proposed in Fig. 3b. The aldehyde groups of GA are activated in the acidic medium of citric acid and react with hydroxyl groups of PVA and ST-GTMAC. 3.3. Swelling degree and solubility One of the most important properties of biodegradable materials is water absorption capacity and swelling degree. Swelling degree can be affected by many factors such as crosslinking density, the hydrate-ability of the materials, the ionic strength and pH value of the media, as well as temperature of the environment [26]. Visual inspection of the present samples showed appreciable volume increase, usually in a few minutes after immersion of sample in the swelling medium that indicates initial rapid water absorption. Fig. 4 shows the effect of ST-GTMAC, GA, and CA on the swelling and solubility behavior of the prepared films. Fig. 4a presents the swelling degree of PVA/ST-GTMAC/CA25%/GA2% crosslinked film versus ST-GTMAC content. As can be seen in this figure, the swelling degree of the film increased with increasing ST-GTMAC content. This increase can be attributed to the improved hydrate-ability of the crosslinked film due to presence of quaternary ammonium salt moiety onto macromolecular chains of starch which has hydrophilic character and increases water absorption. Increasing ST-GTMAC content can also breakdown hydrogen bonding between PVA chains and reduces crystallinity of PVA. Fig. 4a also shows the solubility of the blend film increased with increasing ST-GTMAC content, indicating incomplete network formation. This can be attributed to the less reactivity of hydroxyl groups in ST-GTMAC chains due to spatial hindrances in comparison with the hydroxyl groups of PVA chains. Fig. 4b shows the swelling degree and solubility of PVA/ST-GTMAC10%/CA25% films with different amounts of GA. The blend films without using GA are soluble in water and their swelling behavior was not investigated. The results have revealed a strong influence of chemical crosslinking on the swelling behavior of the film. It was observed that swelling degree of the crosslinked sample decreased from 216% to 100% and 73% when GA content increased from 0.25% to 2% and 3%, respectively. This can be due to formation of more rigid and compact network with increasing content of GA, which reduces number of hydrophilic hydroxyl groups in polymer chains. This is also confirmed by the decrease of solubility with increasing GA content. The GA content could not be increased more than 3 wt% because of formation of a very rigid material which is not suitable for film formation. However, as can be seen in Fig. 4b, the blend film still shows some solubility in water which can be due to unreacted ST-GTMAC trapped inside the network. Other reported studies of PVA based blend materials supported these findings [16,27]. Fig. 4c shows the swelling degree and solubility of PVA/ST-GTMAC10%/GA2% crosslinked films with different citric acid (CA) contents. It has been reported that CA can form crosslinks between starch and PVA macromolecular chains, and as a result of this water absorption of the blend films decreases. According to
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Fig. 3. (a) Photograph images of crosslinked PVA film and crosslinked blend films of PVA containing 10 wt% and 30 wt% ST-GTMAC, (b) crosslink reactions of preparation of film.
Fig. 4c, swelling degree of the crosslinked films decreased and solubility has increased with increasing CA content. These results are well consistent with those reported by other researchers [28–30]. Moreover, addition of CA decreases the pH of the medium leading to more activation of GA for crosslinking reactions.
3.4. Mechanical properties Films for packaging are required to maintain their integrity in order to withstand the stress that occurs during shipping, handling and storage. Tensile strength and elongation at break of crosslinked blend films versus different amounts of ST-GTMAC, GA, and CA are presented in Fig. 5. Elongation at break (El@Br), which is determined at the point where the film breaks under tensile stress, gives information about the film flexibility and stretch ability. As can be
seen in Fig. 5a, elongation at break decreases as the amount of STGTMAC in the crosslinked film increases. The same results have been also reported for PVA/starch blend films by other researchers [1,3]. The tensile strength of the films showed a sharp increase with addition of ST-GTMAC up to 20 wt%, and then started to decrease sharply when the amount of ST-GTMAC increased to 30 wt% and 40 wt%. This behavior proposes the establishment of interchain bonds between PVA and ST-GTMAC which reinforce the cohesion of network structure, thus increasing the tensile strength but restricting movement of polymer chains and reduce film stretch ability. The same results were observed for blend films of PVA/chitosan [2,31]. Fig. 5b shows the effect of GA content on the tensile properties of PVA/ST-GTMAC10%/CA25% films. With addition of 1 wt% GA, tensile strength of the film increased from 47.4 MPa to 56.4 MPa and its elongation at break decreased from 138% to 62% (Fig. 5b). This
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Fig. 4. Swelling degree and solubility of films: (a) effect of ST-GTMAC content (CA: 25 wt% and GA: 2 wt%), (b) effect of GA content (ST-GTMAC: 10 wt%, CA: 25 wt%), (c) effect of CA content (ST-GTMAC: 10 wt%, GA: 2 wt%).
is due to crosslinking and formation of strong network between polymer chains. With increasing the amount of GA up to 3 wt%, the tensile strength increased slowly up to 59.3 MPa while elongation decreased to 53.6%. Therefore, 2 wt% of GA was selected as an optimum amount. It is well known that plasticizer also affect the mechanical properties [28]. As can be seen in Fig. 5c, addition of 10 wt% of CA into PVA/ST-GTMAC10%/GA2% increased tensile strength of the film sharply from 59.0 MPa to 74.5 MPa, this can shows that CA acts as crosslinker at low concentration via formation of hydrogen bonding with hydroxyl and carboxylic acid groups of PVA and ST-GTMAC macromolecular chains. However, when the amount of CA increased from 10 wt% up to 30 wt%, tensile strength decreased and elongation increased, significantly. This can show the plasticization effect of CA at high concentrations which reduced the interactions among the macromolecules, which resulted in the decrease of the tensile strength and increase of the elongation at break. 3.5. Thermal analysis Differential scanning calorimetry (DSC) analysis was used to study thermal properties such as glass transition temperature (Tg ), melting temperature (Tm ) and melting enthalpy (Hm ) of PVA and its film samples with ST-GTMAC/CA/GA. The obtained data
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Fig. 5. Tensile strength (TS) and elongation at break (El@ Br) of films: (a) effect of ST-GTMAC content (CA: 25 wt% and GA: 2 wt%), (b) effect of GA content (ST-GTMAC: 10 wt%, CA: 25 wt%), (c) effect of CA content (ST-GTMAC: 10 wt%, GA: 2 wt%).
are listed in Table 1. As a general rule, any structural feature that reduces segmental mobility of polymer chains or free volume will increase Tg and Tm [2,3]. PVA showed two transitions; a broad endothermic peak in the range of 60–100 ◦ C which can be related to the Tg of the amorphous section and also loss of absorbed moisture, and a sharp endothermic peak at 192 ◦ C due to melting of the crystalline section. Starch is amorphous and hygroscopic in nature and does not have a definite Tg or Tm . The Tg value for ST-GTMAC could not be observed in this work. The absence of a clear Tg may be due to amorphous chains surrounded by crystalline domains, the presence of moisture, physical crosslinks inhibiting the movements of the amorphous chain segments, and the presence of inter-crystalline phases not showing normal thermal behavior [28]. As can be seen in Table 1, blend film of PVA/10 wt%ST-GTMAC showed Tg at lower Table 1 DSC results of PVA and its blend films. Sample
Tg ◦ C
Tm ◦ C
Hm (J g−1 )
PVA PVA/ST-GTMACa PVA/ST-GTMAC/CAb PVA/ST-GTMAC/CA/GAc
76 70 66 70
192 190 176 –
13.86 7.24 3.68 –
Tg , glass transition temperature; Tm , melting temperature; Hm , melting enthalpy. a ST-GTMAC: 10 wt%. b ST-GTMAC: 10 wt%, CA: 25 wt%. c ST-GTMAC: 10 wt%, CA: 25 wt%, GA: 2 wt%.
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Table 2 Thermogravimetric analysis of PVA, ST-GTMAC and their blend films. Sample
IDT (◦ C)a
FDT (◦ C)b
DT at 20% weight loss (◦ C)
DT at 60% weight loss (◦ C)
Char yield at 600 (%)
PVA ST-GTMAC PVA/ST-GTMACc PVA/ST-GTMAC/CAd PVA/ST-GTMAC/CA/GAe
296 224 247 294 303
379 261 368 410 375
286 224 253 266 307
351 254 337 379 404
5 12 5.4 6 10
a b c d e
Initial decomposition temperature of major weight loss. Final decomposition temperature of major weight loss. ST-GTMAC: 10 wt%. ST-GTMAC: 10 wt%, CA: 25 wt%. ST-GTMAC: 10 wt%, CA: 25 wt%, GA: 2 wt%.
temperature with less crystalinity as indicated by melting enthalpy (Hm ) of PVA which has decreased from 13.86 J g−1 to 7.24 J g−1 . These changes can be due to increase in the free volume and discontinuity and disruption of PVA chain interactions in the presence of ST-GTMAC. The plasticizer molecules penetrate inside the polymer matrix and disturb the rigidity arrangement of crystalline PVA and promote internal lubrication and flexible chain sliding [28]. Incorporation of CA (25 wt%) acted as plasticizer and reduced cohesive force between polymer chains and decreased Tg and Tm of the blend films (Table 1). Addition of 2 wt% GA as cross-linker produced an amorphous crosslinked network with Tg at 70 ◦ C. TGA curves of PVA, ST-GTMAC, blend film of PVA/ST-GTMAC and PVA/ST-GTMAC/CA and crosslinked film of PVA/ST-GTMAC/GA are shown in Fig. 6, and the data including initial and final decomposition temperatures (IDT and FDT), decomposition temperatures for 20% and 60% weight loss, and char yield are listed in Table 2. TGA curve of PVA in Fig. 6 shows three-step decompositions: a 10% weight loss in the range of 100–150 ◦ C due to loss of absorbed moisture, a major weight loss of about 65% occurred from about 280 ◦ C to 380 ◦ C due to formation of volatile disintegrated products like water eliminated from PVA chains, and also ∼20% weight loss from ∼400 ◦ C to 480 ◦ C which is mainly caused by heat decomposition of the polymer chains, and the final products are composed of small molecular carbon and hydrocarbon [28]. TGA curve of ST-GTMAC in Fig. 6 shows about 20% weight loss from 50 ◦ C to 180 ◦ C which can be related to the loss of absorbed moisture and a major weight loss of about 60% from 220 ◦ C to 300 ◦ C due to thermal decomposition of polymer chains. The decomposition of PVA/ST-GTMAC blend showed a two-step process. The decomposition curve of this blend has shifted toward lower temperature in comparison with the decomposition curve of PVA. The first step weight loss can be due to decomposition of ST-GTMAC because thermal stability of ST-GTMAC is lower than that of PVA, as shown in Fig. 6. The second step weight loss can be attributed to decomposition of PVA. A major weight loss in TGA curve for PVA/ST-GTMAC10%/CA25% blend started from 294 ◦ C
Fig. 6. TGA of PVA, ST-GTMAC and their blends.
(Table 2). The addition of CA obviously increased the thermal stability and the residual weight percentage at 600 ◦ C in comparison with the PVA/ST-GTMAC blend. This increase in thermal stability can be due to the interaction of CA molecules with PVA and STGTMAC chains through hydrogen bonding. Finally, TGA curve of PVA/ST-GTMAC/CA/GA crosslinked film showed the highest thermal stability, as shown in Fig. 6. This crosslinked blend film showed 55% and 30% weight losses in the range 300–400 ◦ C and 405–500 ◦ C, respectively. 3.6. UV–vis barrier properties It is important to protect some light sensitive materials from the effects of light, especially UV radiation. The UV–vis spectra of PVA and PVA/ST-GTMAC films (crosslinked by 2 wt% GA and plasticized by 25 wt% CA) in the range of 250–700 nm are shown in Fig. 7 and light transmittance (%T) in some wavelength and transparency value at 600 nm are listed in Table 3. The absorption band at about 270–280 nm in the UV spectra of the films is assigned to C O bond, and its intensity increased with increasing ST-GTMAC content which can be due to interaction between ST-GTMAC and PVA chains [20,32]. As shown in Table 3, the light transmittance of crosslinked blend films measured at different wavelengths decreased with increasing ST-GTMAC content. For example, the light transmittance of neat PVA film was ∼91% at 700 nm and decreased to 67.60%, 63.09%, 51.88% and 38.72% by addition of 10, 20, 30 and 40% ST-GTMAC, respectively. Therefore, PVA/ST-GTMAC blend film has also relatively good protection against visible light but transparency of the blend films decreased to some extent by addition of ST-GTMAC content. It is necessary to emphasize that the greater transparency value represents the lower transparency of the film. So, the film with 40 wt% ST-GTMAC showed the highest transparency values at 600 nm (10.32), which
Fig. 7. UV-Vis spectra of PVA, and blend films with different contents of ST-GTMAC (CA: 25 wt% and GA: 2 wt%).
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Table 3 Light transmittance (T%) and transparency value of PVA and PVA/ST-GTMAC crosslinked blend films at 600 nm. Transparency value (STD)a
ST-GTMAC content (wt%)
T % at particular wavelength (nm) 250 nm
350 nm
600 nm
700 nm
0 10 20 30 40
1.74 8.10 2.97 2.25 0.68
64.71 38.01 25.88 21.47 11.96
90.57 67.45 62.81 51.64 38.63
90.99 67.60 63.09 51.88 38.72
a b
0.97 (0.08)b 4.30 (0.43)b 5.17 (0.51)b 7.20 (0.67)b 10.32 (0.82)b
Standard deviation. The significant differences (p < 0.05).
Fig. 8. Antibacterial activity of crosslinked PVA, the PVA/ST-GTMAC/GA and PVA/ST-GTMAC/CA using zone of growth inhibition (a: PVA/GA2%, b: PVA/STGTMAC30%/CA25%/GA2%, c: PVA/ST-GTMAC30%/GA2%).
indicating the lowest transparency of the film. It is obvious that PVA/ST-GTMAC has better barrier properties against UV and could retard photo-oxidation of materials by UV light. 3.7. Antibacterial properties The direct incorporation of antimicrobial agents which are chemically linked with the polymer chains is a convenient methodology by which antimicrobial activity can be achieved. Therefore, antibacterial activity of crosslinked films of PVA/STGTMAC30%/GA2% and PVA/ST-GTMAC30%/CA25%/GA2% were compared with antibacterial activity of crosslinked PVA film against Gram-positive (S. aureus and B. subtilis) and Gram-negative (E. coli and P. aeruginosa) microorganisms. The results are shown in Fig. 8. Antibiotics Gentamicin (10 g disk−1 ) and Chloramphenicol (30 g disk−1 ) were used as standards and the inhibitory effect was measured based on clear zone surrounding circular film disk. Therefore, clear zone diameter included diameter of film disk was measured, and that was always higher than the diameter of film disk whenever clearing zone was present. If there was no clear zone surrounding, it suggested that there is no inhibitory zone, and furthermore, the diameter was valued as zero [29]. As shown in Fig. 8, PVA/GA2% crosslinked film (image a) did not have any antibacterial activity against the tested microorganisms. While crosslinked films of PVA/ST-GTMAC30%/CA25%/GA2% (image b), and PVA/ST-GTMAC30%/GA2% (image c), in Fig. 8, showed fair antibacterial activity against these microorganisms in comparison with the commercial antibiotics of gentamicin and specially chloramphenicol which did not show any inhibitory activity against P. aeruginosa bacteria. The exact mechanism for the antibacterial activity of ST-GTMAC is its positively charged amino groups which interact with negatively charged microbial cell membranes, leading to the leakage of proteinaceous and other intracellular constituents of the microorganisms. Also, as shown in Fig. 8, PVA/ST-GTMAC/CA/GA crosslinked film showed better antibacterial activity against Gram-negative bacteria (E. coli and P. aeruginosa) than crosslinked blend film of PVA/ST-GTMAC/GA. This
can be attributed to the antibacterial activity of CA against these microorganisms. CA is a weak acid and is used as preservative agent in food industries, and its antibacterial activity against Gram-negative bacteria has also been reported previously [33,34]. Therefore, CA acts not only as a plasticizer but also improves the antibacterial activity of the prepared packaging crosslinked films. Therefore, inherent antibacterial properties of positively charged amino groups in ST-GTMAC chains and film forming ability of PVA with appropriate amounts of GA as crosslinker and CA as a plasticizer make the prepared crosslinked film an ideal choice for use as a biodegradable antibacterial packaging material. 4. Conclusion In this study, a novel crosslinked film of modified starch with quaternary ammonium salt (ST-GTMAC) and PVA was prepared in the presence of CA as plasticizer and GA as crosslinking agent. The effect of ST-GTMAC, CA and GA contents on the properties of the crosslinked films such as swelling degree, solubility, mechanical, thermal, barrier against UV radiation, and antibacterial activity was investigated. Results indicated that GA content increased tensile strength, Tg , and decomposition temperature due to chemical crosslink reactions while elongation at break, swelling degree and solubility decreased. A strong film was obtained at low concentration of CA (up to 10%) due to inter- and intra-molecular hydrogen bonding with the polymer chains. CA acted as plasticizer at higher concentrations and reduced tensile strength and Tg of the film. The advantages of using quaternary ammonium modified starch (STGTMAC) to prepare crosslinked film with PVA are: antimicrobial activity against Gram-positive (S. aureus and B. subtilis) and Gramnegative (E. coli and P. aeruginosa) bacteria, the antibacterial agent cannot leached out from the film, and reduction of UV light penetration. Therefore, it can be suggested that prepared crosslinked film from PVA/ST-GTMAC20%/CA25%/GA2% with acceptable mechanical and thermal properties, good transparency and UV-protection, and fair antibacterial activity can be a candidate for potential applications in the packaging industry.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2015. 07.008
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Performance properties and antibacterial activity of crosslinked films of quaternary ammonium modified starch and poly(vinyl alcohol) Zahra Sekhavat Pour, Pooyan Makvandi, Mousa Ghaemy ∗ Polymer Research Laboratory, Department of Chemistry, University of Mazandaran, Babolsar, Iran
a r t i c l e
i n f o
Article history: Received 11 May 2015 Received in revised form 20 June 2015 Accepted 8 July 2015 Available online 15 July 2015 Keywords: Poly(vinyl alcohol) Antibacterial activity Biomaterial Quaternary ammonium starch Chemical crosslinking
a b s t r a c t There has been a growing interest in developing antibacterial polymeric materials. In the present work, novel antibacterial cross-linked blend films were prepared based on polyvinyl alcohol (PVA) and quaternary ammonium starch (ST-GTMAC) using citric acid (CA) as plasticizer and glutaraldehyde (GA) as cross-linker. The ST-GTMAC was successfully synthesized from reaction between water-soluble oxidized starch and glycidyltrimethylammonium chloride (GTMAC). The effect of ST-GTMAC, CA and GA contents on the swelling, solubility, mechanical and thermal properties of the films was investigated. It was found that incorporation of ST-GTMAC reduced UV-transmittance and provided antibacterial properties, increasing GA content increased tensile strength and decreased solubility and swelling degree of the films, while CA acted as plasticizer when its concentration was above 10 wt%. The results showed that ST-GTMAC/PVA/CA/GA film has fair antibacterial activity against Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. These results suggest that the prepared film might be used as potential antibacterial material in medical and packaging applications. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Biodegradable plastics have attracted growing attention because of their potential use in the replacement of traditional petroleum-based synthetic polymers that do not degrade in a land fill and poses a serious environmental threat. Poly(vinyl alcohol) (PVA) is recognized as one of the very few vinyl polymers soluble in water also susceptible of biodegradation in the presence of suitably acclimated microorganisms. However, PVA has poor dimensional stability due to high moisture absorption and relatively high price compared to other commercial polymers. Therefore, increasing attention is devoted to the preparation of environmentally compatible PVA-based materials for a wide range of applications such as wound dressing material, various coatings, and active packaging including controlled delivery systems [1–4]. Among all natural biopolymers, PVA-blended with renewable and abundant polysaccharide such as starch can be most promising because of its easy availability, biodegradability, and lower cost. Starch has been used as a filler to produce reinforced plastics to reduce environmental pollution [5,6]. PVA/starch blend is suitable to be used as
∗ Corresponding author. E-mail address: [email protected] (M. Ghaemy). http://dx.doi.org/10.1016/j.ijbiomac.2015.07.008 0141-8130/© 2015 Elsevier B.V. All rights reserved.
biodegradable packaging and agricultural materials [7]. Their physical properties can be improved by adding plasticizers such as glycerol [7,8], by using chemically modified starch [9,10], by adding crosslinker [3,11], or by preparing biodegradable nanocomposites [12,13]. Antimicrobial packaging has one of the most promising active packaging systems which improve the food safety and shelf-life. There are several ways in which antibacterial properties can be accomplished including direct incorporation of an antimicrobial agent into a polymer, coating or adsorbing an antibacterial agent onto the polymer surface, immobilization of an antibacterial agent in the polymer via ionic or covalent bonding, etc. [1]. The immobilization of quaternary ammonium salt was proved to be an efficient approach to impose antibacterial property on a biomaterial [14,15]. Glycidyltrimethylammonium chloride (GTMAC) is a quaternary ammonium salt that has received much attention as an antibacterial agent [16,17], and its antibacterial activity in chitosan was investigated after preparation of crosslinked blend films with PVA [16]. However, in our knowledge, there has been no study on preparation, characterization and antibacterial activity of crosslinked blend film composed of quaternary ammonium modified starch as a low cost biomaterial and PVA. Therefore, the aim of this work was to study the antimicrobial activity of the crosslinked blend films of PVA with starch chemically bonded with antimicrobial agent,
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glycidyl trimethylammonium chloride (GTMAC). The antimicrobial functionalized starch (ST-GTMAC) was prepared by reacting water soluble oxidized starch with GTMAC, and then ST-GTMAC was blended with PVA, plasticized by citric acid (CA) and chemically crosslinked by glutaraldehyde (GA). The effect of ST-GTMAC, GA and CA contents in the blend films was investigated on the physical properties such as tensile strength, thermal properties, swelling behavior, solubility, and UV–vis protection. Also, antibacterial activity of the films was investigated against Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. 2. Experimental 2.1. Materials Starch, polyvinyl alcohol (PVA, 98% hydrolyzed with a molecular weight average of 49,000), sodium bromate, trimethylamine solution (33 wt% in ethanol), glutaraldehyde (GA) aqueous solution (25 wt%), epichlorohydrin and citric acid (CA) were purchased from Merk (Germany) and used without further purification. 2.2. Oxidation of starch Oxidized starch was synthesized according the procedure described elsewhere [18]. Briefly, 3 g starch, 15 mL distilled water, and 1.05 equivalent NaBrO3 (2.927 g) per glucose unit were mixed in a 500 mL flask. Then, 5.5 mL (1.77 M) H2 SO4 was manually added into the flask within 60 min at room temperature. The reaction was stopped after 24 h by neutralizing the solution with NaOH (1 M) until pH 9. The oxidation product was precipitated by addition of ethanol, and then washed three times with a water-ethanol mixture (30 mL of distilled H2 O and 300 mL of cold ethanol) and dried in a vacuum oven at 30 ◦ C for 24 h. Back titration was used to determine the carboxyl group content of oxidized starch. Starch solution (0.5 g dry oxidized starch in 300 mL distilled water) was adjusted to pH 2.5 with 0.1 M HCl at room temperature, stirred for 15 min and titrated to pH 8.3 with standardized 0.1 M NaOH. Starch was used as blank sample. Carboxyl content of the sample was calculated as follows [18]: Percentage of carboxyl content =
[(Sample-blank) mL × molarity of NaOH × 100 × 0.045] Sample weight in grams
(1)
The content of carboxyl groups of the oxidized starch was about 10.36 ± 0.86% by titration method. 2.3. Preparation of glycidyltrimethylammonium chloride (GTMAC) In a three-necked round bottom flask equipped with a magnetic stir, a condenser, and nitrogen gas inlet tube, 4.0 mmol trimethylamine was added to 12.0 mmol epichlorohydrin in 20 mL ethanol. The solution was then heated to reflux for 24 h. After evaporation of the solvent under reduced pressure, quaternary ammonium salt was purified by recrystallization from acetone. (2,3-epoxypropyl) trimethylammonium chloride (GTMAC) was obtained by raising the pH of quaternary ammonium salt aqueous solution to 8 by using 15% (w/v) NaOH [19]. 2.4. Preparation of (2-hydroxy)propyl-3-trimethylammonium starch chloride (ST-GTMAC) In a three-necked round bottom flask equipped with a magnetic stirer, a condenser, and nitrogen gas inlet tube, 1.5 g oxidized
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starch (ST) was dissolved in distilled water (20 mL) and then 0.5 g GTMAC and catalytic amount of triethylamine solution (25% v/v) were added. The reaction was carried out under nitrogen at 70 ◦ C for 24 h. Then, the solution was poured into acetone/methanol (50/50, v/v) mixture to obtain the precipitate (ST-GTMAC) which was filtered and washed with acetone several times. Finally, ST-GTMAC was extracted with hot methanol in a soxhlet apparatus for 48 h to remove unreacted glycidyltrimethylammonium chloride. 2.5. Preparation of PVA/ST-GTMAC crosslinked film Polymer films were prepared by solution casting method. PVA was dissolved in distilled water (at 5% w/v) in a flask under magnetic stirring at 90 ◦ C for 3 h. ST-GTMAC was added at different mass ratios where part of the PVA was gradually replaced by ST-GTMAC up to 40%. Then citric acid (0–30 wt% based on the weight of polymer blend) as plasticizer was added and the mixture was stirred for 2 h at 40 ◦ C, the temperature was raised to 65 ◦ C and glutaraldehyde (0.25–3 wt% based on the weight of polymer blend) as crosslinking agent was gradually added and the solution was stirred for 15 min. Then the solution was cooled down and poured into the Petri dish to be dried at 35 ◦ C to form the crosslinked PVA/ST-GTMAC blend film. The crosslinked film was extracted with hot methanol in a soxhlet apparatus for 24 h to remove unreacted GA, and dried in a vacuum oven at 60 ◦ C. 2.6. Characterization 2.6.1. FTIR and 1 H NMR Fourier-transform infrared (FT-IR) spectra were recorded on a Bruker Vector 22 FTIR spectrometer in the range of 400–4000 cm−1 . 1 H NMR (400 MHz) spectra were obtained on a Bruker DRX 400 Advance spectrometer at room temperature with D2 O as solvent and tetramethylsilane (TMS) as the internal standard. Chemical shifts are reported in ppm relative to the deuterated solvent resonances. 2.6.2. Swelling degree and solubility of films The percentage of degree of swelling (DS%) and solubility (S%) of the films was measured using the following method. Dried PVA/STGTMAC crosslinked blend films were immersed in distilled water at room temperature. After the equilibrium (24 h), moisture on the surface of the film was removed and the weight of the film was measured. DS of the film was calculated using Eq. (2). DS% =
(We − W0 ) × 100 W0
(2)
where We is the weight of the film at the adsorbing equilibrium, and W0 is the initial dry weight of the film. The swollen film was dried again for 24 h at 30 ◦ C, and its solubility (S) was calculated using Eq. (3). S% =
W0 − Wd × 100 W0
(3)
where W0 represents the initial weight of the specimen and Wd is the weight after drying process. Each experiment was performed in triplicate. 2.6.3. Mechanical properties of films Tensile strength (TS) and elongation at break (El@br) were measured for each crosslinked film by Instron Universal Testing Machine (Model DBBP 500, BONGSHIN). Three rectangular shaped specimens were cut out from each film and each specimen was 12 mm wide with an average thickness of 0.05 mm. The gauge length and grip distance were both 50.0 mm. Crosshead speed was 5 mm min−1 and load cell was 250 kgf.
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Fig. 1. Synthetic approach of quaternary ammonium starch (ST-GTMAC).
2.6.4. Thermal properties The thermal behavior of crosslinked blend films was evaluated by thermal gravimetrical analysis (TGA) (Rheometric Scientific, USA) from room temperature up to 600 ◦ C with the heating rate of 10 ◦ C min−1 . A differential scanning calorimetry (DSC) (DSC1 METTLER TOLEDO, Switzerland) was employed to investigate thermal properties of the crosslinked blend films. The sample (3–10 mg) was placed in an aluminum pan and measurement was carried out under nitrogen flow in the temperature range from 20 to 220 ◦ C at 10 ◦ C min−1 . 2.6.5. Ultraviolet–visible spectrophotometer (UV–vis) UV–vis absorption spectra of the samples (1 × 1 cm2 ) were recorded in the wavelength of 250–700 nm using a Perkin Elmer Instruments (Lambda 35) UV–vis 141 spectrophotometer. The transparency value of the crosslinked films was calculated using Eq. (4) [20]. Transparency = −(log T600 )/x
(4)
where T600 is the fractional transmittance at 600 nm and x is the film thickness (mm). The greater value represents the lower transparence of the film.
2.6.6. Antibacterial activity assay The in vitro antibacterial activities of the compounds were assayed using direct contact test with agar diffusion. The test compounds were prepared as film with a 7 mm in diameter then placed on the surface of inoculated agar plates [21]. The antibacterial activity of the compounds was investigated against four bacterial. Test organisms included E. coli PTCC 1330, P. aeruginosa PTCC 1074, S. aureus ATCC 35923, B. subtilis PTCC 1023. All tested bacteria were maintained in Mueller-Hinton broth (Merck). Before using the cultures, they were standardized with a final cell density of approximately 108 cfu mL−1 . For the direct contact agar diffusion test, the Mueller-Hinton agar plate (Merck) media were used. The agar plates were inoculated from the standardized cultures of the test organisms using a sterile cotton swab then spread as uniformly as possible throughout the entire media. The film was introduced on the upper layer of the seeded agar plate. Mitis-salivaris agar plate was incubated at 37 ◦ C for 72–96 h supplied with 5% CO2 , SABOURAUD Dextrose agar plate and Mueller-Hinton agar plate were incubated at 37 ◦ C for 24–48 h. The antibacterial activities of the compounds were compared with known antibiotic gentamicin (10 g disk−1 ), chloramphenicol (30 g disk−1 ). Positive control plates were streaked with test organisms, but no film was
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used. Antibacterial activity was evaluated by measuring the diameter of inhibition zone (mm) on the surface of plates and the results were reported as mean ± SD after three repeats.
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are assigned to methine proton (b), methylene protons (c), and methylene protons (a), respectively [25], as indicated in Fig. 1S. 3.2. Appearance of PVA/ST-GTMAC crosslinked films
3. Results and discussion 3.1. Synthesis and characterization of ST-GTMAC The synthetic approach for the preparation of ST-GTMAC is illustrated in Fig. 1. Introduction of carboxylic groups (10.36 ± 0.86%) in the oxidized starch structure made starch to dissolve in water completely. In quaternization reaction, the hydroxyl and carboxylic acid groups of the oxidized starch can react with the epoxide ring of glycidyltrimethylammonium chloride (GTMAC) leading to grafting of quaternary ammonium salt moiety onto macromolecular chains of starch to give ST-GTMAC. FT-IR spectra of starch, oxidized starch and ST-GTMAC are shown in Fig. 2. In the spectrum of starch, the absorption bands in the region of 700–1000 cm−1 are due to atomic group involved in the anomeric form [22], in the region of 1000–1200 cm−1 are due to stretching vibration of C O, and in the region of 1200–1500 cm−1 are related to C H bending [23]. The absorption bands in regions of 2800–3000 cm−1 and 3300–3500 cm−1 are assigned to C H and O H stretching, respectively. In the spectrum of oxidized starch, two strong absorption bands are observed at 1613 and 1419 cm−1 due to carboxylate group which is due to oxidation of starch [18,24]. In the FT-IR spectrum of GTMAC, the absorption bands at around 948 cm−1 are the characteristic of epoxide group. Absorption bands in the region of 600–1000 cm−1 are due to stretching vibrations of the NR4 + complexes. The main absorption band of GTMAC is at 1480 cm−1 due to C H symmetric bending of the methyl groups on the quaternary ammonium substituent [17,25]. FT-IR spectrum of ST-GTMAC is similar to that of oxidized starch except for the presence of an absorption band at 1480 cm−1 due to methyl groups on the quaternary ammonium substituent. These results confirm that quaternary ammonium moieties have been chemically attached to the macromolecular chains of starch. In the 1 H NMR spectrum of oxidized starch (Fig. 1S), signals were observed at 5.48 ppm related to the anomeric proton of anhydroglucose unit, other anomeric protons were appeared at 4.8–5.54 ppm, and signals of ring protons were observed in the range of 3.5–4.19 [18]. The characteristic signal of ST-GTMAC was appeared at 3.18 ppm, which is related to the protons of trimethylammonium group (d) [17]. Other signals at 4.22, 3.45 and 2.2 ppm
Fig. 2. FT-IR spectrum of starch, oxidized starch, GTMAC and ST-GTMAC.
Preparation of PVA/ST-GTMAC crosslinked films was carried out by mixing a solution of different weight ratios of PVA, STGTMAC, citric acid (CA) as plasticizer, and glutaraldehyde (GA) as crosslinking agent. Then clear films were prepared by solution casting method. Fig. 3a shows transparency of PVA film, PVA/STGTMAC10%/GA2%/CA25%, and PVA/ST-GTMAC30%/GA2%/CA25% crosslinked films. As can be seen in Fig. 3a, PVA film is colorless and transparent but incorporation of ST-GTMAC up to 30 wt% has changed the color of the film slightly to yellowish. Both PVA and ST-GTMAC macromolecular chains are involved in the crosslink reactions with GA, as proposed in Fig. 3b. The aldehyde groups of GA are activated in the acidic medium of citric acid and react with hydroxyl groups of PVA and ST-GTMAC. 3.3. Swelling degree and solubility One of the most important properties of biodegradable materials is water absorption capacity and swelling degree. Swelling degree can be affected by many factors such as crosslinking density, the hydrate-ability of the materials, the ionic strength and pH value of the media, as well as temperature of the environment [26]. Visual inspection of the present samples showed appreciable volume increase, usually in a few minutes after immersion of sample in the swelling medium that indicates initial rapid water absorption. Fig. 4 shows the effect of ST-GTMAC, GA, and CA on the swelling and solubility behavior of the prepared films. Fig. 4a presents the swelling degree of PVA/ST-GTMAC/CA25%/GA2% crosslinked film versus ST-GTMAC content. As can be seen in this figure, the swelling degree of the film increased with increasing ST-GTMAC content. This increase can be attributed to the improved hydrate-ability of the crosslinked film due to presence of quaternary ammonium salt moiety onto macromolecular chains of starch which has hydrophilic character and increases water absorption. Increasing ST-GTMAC content can also breakdown hydrogen bonding between PVA chains and reduces crystallinity of PVA. Fig. 4a also shows the solubility of the blend film increased with increasing ST-GTMAC content, indicating incomplete network formation. This can be attributed to the less reactivity of hydroxyl groups in ST-GTMAC chains due to spatial hindrances in comparison with the hydroxyl groups of PVA chains. Fig. 4b shows the swelling degree and solubility of PVA/ST-GTMAC10%/CA25% films with different amounts of GA. The blend films without using GA are soluble in water and their swelling behavior was not investigated. The results have revealed a strong influence of chemical crosslinking on the swelling behavior of the film. It was observed that swelling degree of the crosslinked sample decreased from 216% to 100% and 73% when GA content increased from 0.25% to 2% and 3%, respectively. This can be due to formation of more rigid and compact network with increasing content of GA, which reduces number of hydrophilic hydroxyl groups in polymer chains. This is also confirmed by the decrease of solubility with increasing GA content. The GA content could not be increased more than 3 wt% because of formation of a very rigid material which is not suitable for film formation. However, as can be seen in Fig. 4b, the blend film still shows some solubility in water which can be due to unreacted ST-GTMAC trapped inside the network. Other reported studies of PVA based blend materials supported these findings [16,27]. Fig. 4c shows the swelling degree and solubility of PVA/ST-GTMAC10%/GA2% crosslinked films with different citric acid (CA) contents. It has been reported that CA can form crosslinks between starch and PVA macromolecular chains, and as a result of this water absorption of the blend films decreases. According to
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Fig. 3. (a) Photograph images of crosslinked PVA film and crosslinked blend films of PVA containing 10 wt% and 30 wt% ST-GTMAC, (b) crosslink reactions of preparation of film.
Fig. 4c, swelling degree of the crosslinked films decreased and solubility has increased with increasing CA content. These results are well consistent with those reported by other researchers [28–30]. Moreover, addition of CA decreases the pH of the medium leading to more activation of GA for crosslinking reactions.
3.4. Mechanical properties Films for packaging are required to maintain their integrity in order to withstand the stress that occurs during shipping, handling and storage. Tensile strength and elongation at break of crosslinked blend films versus different amounts of ST-GTMAC, GA, and CA are presented in Fig. 5. Elongation at break (El@Br), which is determined at the point where the film breaks under tensile stress, gives information about the film flexibility and stretch ability. As can be
seen in Fig. 5a, elongation at break decreases as the amount of STGTMAC in the crosslinked film increases. The same results have been also reported for PVA/starch blend films by other researchers [1,3]. The tensile strength of the films showed a sharp increase with addition of ST-GTMAC up to 20 wt%, and then started to decrease sharply when the amount of ST-GTMAC increased to 30 wt% and 40 wt%. This behavior proposes the establishment of interchain bonds between PVA and ST-GTMAC which reinforce the cohesion of network structure, thus increasing the tensile strength but restricting movement of polymer chains and reduce film stretch ability. The same results were observed for blend films of PVA/chitosan [2,31]. Fig. 5b shows the effect of GA content on the tensile properties of PVA/ST-GTMAC10%/CA25% films. With addition of 1 wt% GA, tensile strength of the film increased from 47.4 MPa to 56.4 MPa and its elongation at break decreased from 138% to 62% (Fig. 5b). This
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Fig. 4. Swelling degree and solubility of films: (a) effect of ST-GTMAC content (CA: 25 wt% and GA: 2 wt%), (b) effect of GA content (ST-GTMAC: 10 wt%, CA: 25 wt%), (c) effect of CA content (ST-GTMAC: 10 wt%, GA: 2 wt%).
is due to crosslinking and formation of strong network between polymer chains. With increasing the amount of GA up to 3 wt%, the tensile strength increased slowly up to 59.3 MPa while elongation decreased to 53.6%. Therefore, 2 wt% of GA was selected as an optimum amount. It is well known that plasticizer also affect the mechanical properties [28]. As can be seen in Fig. 5c, addition of 10 wt% of CA into PVA/ST-GTMAC10%/GA2% increased tensile strength of the film sharply from 59.0 MPa to 74.5 MPa, this can shows that CA acts as crosslinker at low concentration via formation of hydrogen bonding with hydroxyl and carboxylic acid groups of PVA and ST-GTMAC macromolecular chains. However, when the amount of CA increased from 10 wt% up to 30 wt%, tensile strength decreased and elongation increased, significantly. This can show the plasticization effect of CA at high concentrations which reduced the interactions among the macromolecules, which resulted in the decrease of the tensile strength and increase of the elongation at break. 3.5. Thermal analysis Differential scanning calorimetry (DSC) analysis was used to study thermal properties such as glass transition temperature (Tg ), melting temperature (Tm ) and melting enthalpy (Hm ) of PVA and its film samples with ST-GTMAC/CA/GA. The obtained data
601
Fig. 5. Tensile strength (TS) and elongation at break (El@ Br) of films: (a) effect of ST-GTMAC content (CA: 25 wt% and GA: 2 wt%), (b) effect of GA content (ST-GTMAC: 10 wt%, CA: 25 wt%), (c) effect of CA content (ST-GTMAC: 10 wt%, GA: 2 wt%).
are listed in Table 1. As a general rule, any structural feature that reduces segmental mobility of polymer chains or free volume will increase Tg and Tm [2,3]. PVA showed two transitions; a broad endothermic peak in the range of 60–100 ◦ C which can be related to the Tg of the amorphous section and also loss of absorbed moisture, and a sharp endothermic peak at 192 ◦ C due to melting of the crystalline section. Starch is amorphous and hygroscopic in nature and does not have a definite Tg or Tm . The Tg value for ST-GTMAC could not be observed in this work. The absence of a clear Tg may be due to amorphous chains surrounded by crystalline domains, the presence of moisture, physical crosslinks inhibiting the movements of the amorphous chain segments, and the presence of inter-crystalline phases not showing normal thermal behavior [28]. As can be seen in Table 1, blend film of PVA/10 wt%ST-GTMAC showed Tg at lower Table 1 DSC results of PVA and its blend films. Sample
Tg ◦ C
Tm ◦ C
Hm (J g−1 )
PVA PVA/ST-GTMACa PVA/ST-GTMAC/CAb PVA/ST-GTMAC/CA/GAc
76 70 66 70
192 190 176 –
13.86 7.24 3.68 –
Tg , glass transition temperature; Tm , melting temperature; Hm , melting enthalpy. a ST-GTMAC: 10 wt%. b ST-GTMAC: 10 wt%, CA: 25 wt%. c ST-GTMAC: 10 wt%, CA: 25 wt%, GA: 2 wt%.
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Table 2 Thermogravimetric analysis of PVA, ST-GTMAC and their blend films. Sample
IDT (◦ C)a
FDT (◦ C)b
DT at 20% weight loss (◦ C)
DT at 60% weight loss (◦ C)
Char yield at 600 (%)
PVA ST-GTMAC PVA/ST-GTMACc PVA/ST-GTMAC/CAd PVA/ST-GTMAC/CA/GAe
296 224 247 294 303
379 261 368 410 375
286 224 253 266 307
351 254 337 379 404
5 12 5.4 6 10
a b c d e
Initial decomposition temperature of major weight loss. Final decomposition temperature of major weight loss. ST-GTMAC: 10 wt%. ST-GTMAC: 10 wt%, CA: 25 wt%. ST-GTMAC: 10 wt%, CA: 25 wt%, GA: 2 wt%.
temperature with less crystalinity as indicated by melting enthalpy (Hm ) of PVA which has decreased from 13.86 J g−1 to 7.24 J g−1 . These changes can be due to increase in the free volume and discontinuity and disruption of PVA chain interactions in the presence of ST-GTMAC. The plasticizer molecules penetrate inside the polymer matrix and disturb the rigidity arrangement of crystalline PVA and promote internal lubrication and flexible chain sliding [28]. Incorporation of CA (25 wt%) acted as plasticizer and reduced cohesive force between polymer chains and decreased Tg and Tm of the blend films (Table 1). Addition of 2 wt% GA as cross-linker produced an amorphous crosslinked network with Tg at 70 ◦ C. TGA curves of PVA, ST-GTMAC, blend film of PVA/ST-GTMAC and PVA/ST-GTMAC/CA and crosslinked film of PVA/ST-GTMAC/GA are shown in Fig. 6, and the data including initial and final decomposition temperatures (IDT and FDT), decomposition temperatures for 20% and 60% weight loss, and char yield are listed in Table 2. TGA curve of PVA in Fig. 6 shows three-step decompositions: a 10% weight loss in the range of 100–150 ◦ C due to loss of absorbed moisture, a major weight loss of about 65% occurred from about 280 ◦ C to 380 ◦ C due to formation of volatile disintegrated products like water eliminated from PVA chains, and also ∼20% weight loss from ∼400 ◦ C to 480 ◦ C which is mainly caused by heat decomposition of the polymer chains, and the final products are composed of small molecular carbon and hydrocarbon [28]. TGA curve of ST-GTMAC in Fig. 6 shows about 20% weight loss from 50 ◦ C to 180 ◦ C which can be related to the loss of absorbed moisture and a major weight loss of about 60% from 220 ◦ C to 300 ◦ C due to thermal decomposition of polymer chains. The decomposition of PVA/ST-GTMAC blend showed a two-step process. The decomposition curve of this blend has shifted toward lower temperature in comparison with the decomposition curve of PVA. The first step weight loss can be due to decomposition of ST-GTMAC because thermal stability of ST-GTMAC is lower than that of PVA, as shown in Fig. 6. The second step weight loss can be attributed to decomposition of PVA. A major weight loss in TGA curve for PVA/ST-GTMAC10%/CA25% blend started from 294 ◦ C
Fig. 6. TGA of PVA, ST-GTMAC and their blends.
(Table 2). The addition of CA obviously increased the thermal stability and the residual weight percentage at 600 ◦ C in comparison with the PVA/ST-GTMAC blend. This increase in thermal stability can be due to the interaction of CA molecules with PVA and STGTMAC chains through hydrogen bonding. Finally, TGA curve of PVA/ST-GTMAC/CA/GA crosslinked film showed the highest thermal stability, as shown in Fig. 6. This crosslinked blend film showed 55% and 30% weight losses in the range 300–400 ◦ C and 405–500 ◦ C, respectively. 3.6. UV–vis barrier properties It is important to protect some light sensitive materials from the effects of light, especially UV radiation. The UV–vis spectra of PVA and PVA/ST-GTMAC films (crosslinked by 2 wt% GA and plasticized by 25 wt% CA) in the range of 250–700 nm are shown in Fig. 7 and light transmittance (%T) in some wavelength and transparency value at 600 nm are listed in Table 3. The absorption band at about 270–280 nm in the UV spectra of the films is assigned to C O bond, and its intensity increased with increasing ST-GTMAC content which can be due to interaction between ST-GTMAC and PVA chains [20,32]. As shown in Table 3, the light transmittance of crosslinked blend films measured at different wavelengths decreased with increasing ST-GTMAC content. For example, the light transmittance of neat PVA film was ∼91% at 700 nm and decreased to 67.60%, 63.09%, 51.88% and 38.72% by addition of 10, 20, 30 and 40% ST-GTMAC, respectively. Therefore, PVA/ST-GTMAC blend film has also relatively good protection against visible light but transparency of the blend films decreased to some extent by addition of ST-GTMAC content. It is necessary to emphasize that the greater transparency value represents the lower transparency of the film. So, the film with 40 wt% ST-GTMAC showed the highest transparency values at 600 nm (10.32), which
Fig. 7. UV-Vis spectra of PVA, and blend films with different contents of ST-GTMAC (CA: 25 wt% and GA: 2 wt%).
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Table 3 Light transmittance (T%) and transparency value of PVA and PVA/ST-GTMAC crosslinked blend films at 600 nm. Transparency value (STD)a
ST-GTMAC content (wt%)
T % at particular wavelength (nm) 250 nm
350 nm
600 nm
700 nm
0 10 20 30 40
1.74 8.10 2.97 2.25 0.68
64.71 38.01 25.88 21.47 11.96
90.57 67.45 62.81 51.64 38.63
90.99 67.60 63.09 51.88 38.72
a b
0.97 (0.08)b 4.30 (0.43)b 5.17 (0.51)b 7.20 (0.67)b 10.32 (0.82)b
Standard deviation. The significant differences (p < 0.05).
Fig. 8. Antibacterial activity of crosslinked PVA, the PVA/ST-GTMAC/GA and PVA/ST-GTMAC/CA using zone of growth inhibition (a: PVA/GA2%, b: PVA/STGTMAC30%/CA25%/GA2%, c: PVA/ST-GTMAC30%/GA2%).
indicating the lowest transparency of the film. It is obvious that PVA/ST-GTMAC has better barrier properties against UV and could retard photo-oxidation of materials by UV light. 3.7. Antibacterial properties The direct incorporation of antimicrobial agents which are chemically linked with the polymer chains is a convenient methodology by which antimicrobial activity can be achieved. Therefore, antibacterial activity of crosslinked films of PVA/STGTMAC30%/GA2% and PVA/ST-GTMAC30%/CA25%/GA2% were compared with antibacterial activity of crosslinked PVA film against Gram-positive (S. aureus and B. subtilis) and Gram-negative (E. coli and P. aeruginosa) microorganisms. The results are shown in Fig. 8. Antibiotics Gentamicin (10 g disk−1 ) and Chloramphenicol (30 g disk−1 ) were used as standards and the inhibitory effect was measured based on clear zone surrounding circular film disk. Therefore, clear zone diameter included diameter of film disk was measured, and that was always higher than the diameter of film disk whenever clearing zone was present. If there was no clear zone surrounding, it suggested that there is no inhibitory zone, and furthermore, the diameter was valued as zero [29]. As shown in Fig. 8, PVA/GA2% crosslinked film (image a) did not have any antibacterial activity against the tested microorganisms. While crosslinked films of PVA/ST-GTMAC30%/CA25%/GA2% (image b), and PVA/ST-GTMAC30%/GA2% (image c), in Fig. 8, showed fair antibacterial activity against these microorganisms in comparison with the commercial antibiotics of gentamicin and specially chloramphenicol which did not show any inhibitory activity against P. aeruginosa bacteria. The exact mechanism for the antibacterial activity of ST-GTMAC is its positively charged amino groups which interact with negatively charged microbial cell membranes, leading to the leakage of proteinaceous and other intracellular constituents of the microorganisms. Also, as shown in Fig. 8, PVA/ST-GTMAC/CA/GA crosslinked film showed better antibacterial activity against Gram-negative bacteria (E. coli and P. aeruginosa) than crosslinked blend film of PVA/ST-GTMAC/GA. This
can be attributed to the antibacterial activity of CA against these microorganisms. CA is a weak acid and is used as preservative agent in food industries, and its antibacterial activity against Gram-negative bacteria has also been reported previously [33,34]. Therefore, CA acts not only as a plasticizer but also improves the antibacterial activity of the prepared packaging crosslinked films. Therefore, inherent antibacterial properties of positively charged amino groups in ST-GTMAC chains and film forming ability of PVA with appropriate amounts of GA as crosslinker and CA as a plasticizer make the prepared crosslinked film an ideal choice for use as a biodegradable antibacterial packaging material. 4. Conclusion In this study, a novel crosslinked film of modified starch with quaternary ammonium salt (ST-GTMAC) and PVA was prepared in the presence of CA as plasticizer and GA as crosslinking agent. The effect of ST-GTMAC, CA and GA contents on the properties of the crosslinked films such as swelling degree, solubility, mechanical, thermal, barrier against UV radiation, and antibacterial activity was investigated. Results indicated that GA content increased tensile strength, Tg , and decomposition temperature due to chemical crosslink reactions while elongation at break, swelling degree and solubility decreased. A strong film was obtained at low concentration of CA (up to 10%) due to inter- and intra-molecular hydrogen bonding with the polymer chains. CA acted as plasticizer at higher concentrations and reduced tensile strength and Tg of the film. The advantages of using quaternary ammonium modified starch (STGTMAC) to prepare crosslinked film with PVA are: antimicrobial activity against Gram-positive (S. aureus and B. subtilis) and Gramnegative (E. coli and P. aeruginosa) bacteria, the antibacterial agent cannot leached out from the film, and reduction of UV light penetration. Therefore, it can be suggested that prepared crosslinked film from PVA/ST-GTMAC20%/CA25%/GA2% with acceptable mechanical and thermal properties, good transparency and UV-protection, and fair antibacterial activity can be a candidate for potential applications in the packaging industry.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2015. 07.008
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