silver nanoparticles composite films with UV-light barrier and properties

International Journal of Biological Macromolecules 92 (2016) 842–849

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Preparation of pectin/silver nanoparticles composite films with UV-light barrier and properties Shiv Shankar a , Nattareya Tanomrod b , Saroat Rawdkuen b , Jong-Whan Rhim a,∗ a Department of Food Engineering and Bionanocomposite Research Institute, Mokpo National University, 61 Dorimri, Chungkyemyon, Muangun, 534729 Jeonnam, Republic of Korea, Republic of Korea b Food Technology Program, School of Agro-Industry, Mae Fah Luang University, 333 Moo 1, Thasud, Muang, Chiang Rai 57100, Thailand

a r t i c l e

i n f o

Article history: Received 7 June 2016 Received in revised form 15 July 2016 Accepted 31 July 2016 Available online 1 August 2016 Keywords: Silver nanoparticles Pectin composite film Antibacterial activity

a b s t r a c t Silver nanoparticles (AgNPs) was synthesized by a green method using an aqueous extract of Caesalpinia mimosoides Lamk (CMLE) as reducing and stabilizing agents, and they were used for the preparation of pectin-based antimicrobial composite films. The AgNPs were spherical in shape with the size in the range of 20–80 nm and showed the absorption peak around 500 nm. The pectin/AgNPs composite film exhibited characteristic absorption peak of AgNPs at 480 nm. The surface color and light transmittance of the pectin films were greatly influenced by the addition of AgNPs. The lightness of the films decreased, however, redness and yellowness of the films increased after incorporation of AgNPs. UV-light barrier property of the pectin film increased significantly with a little decrease in the transparency. Though there were no structural changes in the pectin film by the incorporation of CMLE and AgNPs as indicated by the FTIR results, the film properties such as thermal stability, mechanical strength, and water vapor barrier properties of the pectin films increased. The pectin/AgNPs nanocomposite films exhibited strong antibacterial activity against food-borne pathogenic bacteria, Escherichia coli and Listeria monocytogenes. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Recently, demand for the packaging materials made of biopolymer materials has been greatly increased due to the non-biodegradable petroleum-based plastic materials and environmental concerns raised by the discarded plastic materials [1]. Various types of biopolymers from natural resources have been used for the development of biodegradable food packaging or edible films since they are abundant, renewable, environmentallyfriendly, and sustainable [1]. In addition to the good film forming property and comparably high mechanical strength, additional potential functional properties such as antimicrobial, antioxidant, and UV barrier properties of the biopolymer-based packaging materials have provided additional benefits for extending the shelflife and maintaining food quality of packaged foods. As one of such biopolymers, carbohydrate-based biopolymers attracted attention for the preparation of innovative food packaging materials because of their good film forming properties, sustainability, and abundance [2]. Among the carbohydrate polymers, pectin has attracted attention for food packaging and other value added applications due

∗ Corresponding author. E-mail addresses: [email protected], [email protected] (J.-W. Rhim). http://dx.doi.org/10.1016/j.ijbiomac.2016.07.107 0141-8130/© 2016 Elsevier B.V. All rights reserved.

to the environmental friendly nature, good processing ability, and acceptable mechanical and barrier properties. Pectin is an important component of the cell walls of terrestrial plants that widely used as a functional food ingredient, and about 4–5 g of pectin is consumed daily in the diet [3]. Pectin is a family of polysaccharides and oligosaccharides with diverse structures [4]. Since pectin is rich in galacturonic acid (GalA), the FAO and EU stipulated that ‘pectin’ must contain at least 65% of GalA. Pectin can form a gel when homogalacturonan is crosslinked to form a three-dimensional crystalline network in which water and solutes are trapped. Gelling properties of pectin are determined by various factors such as the type of pectin, the degree of methyl esterification, the degree of acetylation, temperature, pH, sugar and, calcium [3]. The poor mechanical and water vapor barrier properties of biopolymers film limited their utilization in a packaging application. Therefore, the research works have been focused on the improvement of such properties by reinforcing biopolymers films with nanofillers such as nano clay, nanometals, and crystalline nanocellulose [5–7]. Some plant extract has also been used to improve film properties by inducing cross-linking between polymer strands and providing UV light screening and antimicrobial functions. Various biopolymer films with such functional properties have been used for active food packaging materials for the protection of packaged foods against microbial contamination and

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extending the shelf-life of packaged food [8]. Among nanofillers, silver nanoparticles (AgNPs) have drawn much attention in food packaging and biomedical applications due to their unique antimicrobial activity against a broad range of micro-organisms and heat stability [9]. The antimicrobial activity of AgNPs depends on the size, shape, stabilizing agents, and preparation methods [9]. AgNPs are synthesized using physical, chemical, and biological methods, in which the biological method is considered as convenient and environmentally friendly [6]. As one of the biological methods, green synthesis of AgNPs using plant extracts is well documented for the large scale production of AgNPs [10]. The present study was aimed at the green synthesis of AgNPs using an aqueous extract of Caesalpinia mimosoides Lamk. To the best of our knowledge, reports on the biological synthesis of nanoparticles using Caesalpinia mimosoides Lamk extract are not available in the literature. Caesalpinia mimosoides Lamk., a native plant of Thailand and locally called Cha-Lueat in Thai, is climbing or erect shrub. Leaves and young shoots of the plant are usually consumed as a fresh vegetable or appetizer. The Caesalpinia mimosoides Lamk. extract (CMLE) is rich in vitamins, carotenoids, and phenolic compounds [11]. The CMLE has been reported to contain various functional materials such as gallic acid, steroids, flavonoids, terpenoids, glycosides, and tannins [12]. This plant showed moderate antioxidant activity with high tannin and total phenolic contents, which led us to examine its potential as reducing agents for the synthesis of silver nanoparticles. The main objective of the present study was to prepare biodegradable functional bionanocomposite films by blending with pectin and silver nanoparticles prepared using edible plant extract. The improved film properties such as mechanical and water vapor barrier properties as well as an additional UV light barrier and antimicrobial activity of the composite films were evaluated. This is the first report on the synthesis of AgNPs using CML extract and preparation of pectin-based composite films with AgNPs for the use of antimicrobial and UV screening active food packaging.

2. Materials and methods 2.1. Materials All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Caesalpinia minosoides Lamk. was obtained from the local fresh market near Mae Fah Luang University (Thasud, Chiang Rai, Thailand). Pectin powder (degree of methyl esterification >50) produced from citrus peel was purchased from CP Kelco Aps (Lille Skonaved, Denmark). Brain heart infusion broth (BHI), tryptic soy broth (TSB), and agar powder were purchased from Duksan Pure Chemicals Co., Ltd. (Ansan, Gyunggido, Korea). Foodborne pathogenic microorganisms, Escherichia coli O157: H7 ATCC 43895 and Listeria monocytogenes ATCC 15313 were obtained from the Korean Collection for Type Culture (KCTC, Seoul, Korea). The bacterial strains were grown in TSB and BHI agar medium and stored at 4 ◦ C for further test.

2.2. Preparation of plant extraction Washed and dewatered shoot tip of CML sample was quick frozen by dipping into liquid nitrogen and powdered by using a blender (Model 1G909, Hamilton beach/Proctor-sllex Inc., Southern Pines, NC, USA) and kept at −20 ◦ C before use. The CML powder was suspended in distilled water with the ratio of 1:20 and microwave assisted extraction was used at 500 W for 32 s (MS2029GW, LG Electronics Co., Ltd, Thailand) following the method of Dahmoune et al., [13]. The extract was filtered through Whatman No.1 filter paper

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and freeze-dried the filtrate. The dried powder was collected into a zip lock PE bag and kept in a desiccator until use. 2.3. Preparation of silver nanoparticles Silver nanoparticles were synthesized using the method of Song & Kim with slight modification [14]. For this, 50 mg of plant extract powder was dissolved in 500 mL of distilled water with vigorous mixing using a magnetic stirrer for 30 min and heated the solution to 80 ◦ C. Five mL of 100 mM aqueous solution of silver nitrate (final concentration 1 mM) was added dropwise to the above solution and mixed continuously until the color of the solution changed. The sample was taken out at a different time interval, and absorbance was measured using a UV–vis spectrophotometer (Mecasys Optizen POP Series UV/Vis, Seoul, Korea) in the range of 200–700 nm. The shape and size of AgNPs were evaluated by transmission electron microscopy (TEM) using the JEOL-2010 instrument. For the TEM observation, suspension of AgNPs was dropped on a carboncoated copper grid, air dried, and image analysis was performed. 2.4. Preparation of pectin/AgNPs nanocomposite film Pectin, pectin/CMLE, and pectin/AgNPs composite films were prepared by the solution casting method. For the preparation of pectin/AgNPs nanocomposite film solution, 4 g of pectin was added slowly into 150 mL of AgNPs suspension (silver concentration ∼100 ␮g/mL), then 2 g of glycerol (50 wt% of pectin) was added as a plasticizer, and heated the mixture at 80 ◦ C for 30 min with stirring. For the preparation of pectin/CMLE film solution, 15 mg of CMLE was dissolved in 150 mL of distilled water and followed by the addition of 4 g of pectin and 2 g glycerol; then the mixture was heated at 80 ◦ C for 30 min. Additionally, the neat pectin film solution was prepared using the same method without the addition of CMLE or AgNPs. The film-forming solution (150 mL) was cast evenly on a leveled Teflon film (Cole-Parmer Instrument Co., Chicago, IL, USA) coated glass plate (24 × 30 cm). The films were dried completely at room temperature for about 48 h. The films were peeled off from the glass plates after drying and preconditioned in a constant humidity and temperature chamber (model FX 1077, Jeio Tech Co. Ltd., Ansan, Korea) controlled at 25 ◦ C and 50% relative humidity (RH) for 48 h. 2.5. Characterization of pectin-based composite films 2.5.1. Morphological observation and FT-IR The surface morphology of the composite films was analyzed by the field emission scanning electron microscopy (FE-SEM, S-4800, Hitachi Co., Ltd., Matsuda, Japan) at an accelerating voltage of 5.0 kV. Fourier transform infrared (FT-IR) spectra of the films were measured by an attenuated total reflectance-Fourier transform infrared (AT-FTIR) spectrophotometer (TENSOR 37 spectrophotometer with OPUS 6.0 software, Billerica, MA, USA) operated at a resolution of 4 cm−1 . 2.5.2. Surface color and optical properties Surface color of the films was evaluated by measuring Hunter L, a, and b color values as well as the total color difference (E) using a Chroma meter (Konica Minolta, CR-400, Tokyo, Japan). Hunter color values (L, a, and b) were determined by taking an average of five readings from each film sample. Total color difference (E) was calculated as follows: E = [(L)2 + (a)2 + (b)2 ]0.5 where L, a, and b were the difference between each color value of standard color plate and film samples, respectively.

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Absorbance and transmittance of the film samples were measured using a UV–vis spectrophotometer (Model 8451A, Hewlett-Packard Co., Santa Alara, CA, USA). The absorption spectrum of the film samples was measured at the wavelength of 200–700 nm. UV-screening and transparency properties of the films were evaluated by measuring the percent transmittance at 280 nm (T280 ) and 660 nm (T660 ), respectively.

Castle, DE, USA). About 5 mg of each film sample was taken in a standard aluminum pan and heated at 30 ◦ C–600 ◦ C at a heating rate of 10 ◦ C/min under a nitrogen flow of 50 cm3 /min. The empty pan was used as a reference. The derivative of TGA (DTG) was calculated using a central finite difference method, and the char content of the samples at 600 ◦ C was determined from the TGA curve [16]. 2.6. Antibacterial activity

2.5.3. Tensile properties The thickness of the films was measured using a hand-held micrometer with an accuracy of 0.001 mm (Digimatic Micrometer, QuantuMike IP 65, Mitutoyo, Japan). The tensile strength (TS), elastic modulus (E), and percent elongation at break (EAB) of the films were determined using an Instron Universal Testing Machine (Model 5565, Instron Engineering Corporation, Canton, MA, USA) according to ASTM D 882-88 standard method. For this, each film was cut into rectangular strips (2.54 cm × 15 cm) using a precision double blade cutter (Model LB.02/A, Metrotech, SA, San Sebastain, Spain). The machine was operated in tensile mode with an initial grip separation of 50 mm and crosshead speed of 50 mm/min. The TS was expressed as MPa, and it was determined by dividing the maximum load (N) by the initial cross-sectional area (m2 ) of the films. The EAB was expressed as %, and it was determined by dividing the extension at the rupture of the film by the initial length of the film (50 mm) multiplied by 100, and the E (MPa) was determined from the slope of the linear portion of the stress-strain curve, which corresponds to the stress divided by the strain of the film sample. Ten measurements were carried out for each film and the average values were presented. 2.5.4. Water vapor permeability, water contact angle, and moisture content The water vapor permeability (WVP) of the film samples was determined using a WVP cup method following the standard method of ASTM E96-95 with modification [15]. The film sample (7.5 cm × 7.5 cm) was mounted on the top of WVP cup (2.5 cm in depth and 6.8 cm in diameter) containing 18 mL of water and sealed tightly to prevent leakage of water vapor. The assembled WVP cup was placed in a humidity chamber (model FX 1077, Jeio Tech Co. Ltd., Ansan, Korea) controlled at 50% RH and 25 ◦ C with an air movement of 198 m/min. The weight loss of the cup was measured every hour for 8 h to determine the water vapor transmission rate (WVTR) (g/m2 s) of the film, then the WVP of the film was calculated in g m/m2 Pa s as follows:

E. coli and L. monocytogenes were used to test the antibacterial activity of the film by the total colony count method [17]. The bacterial colony was incubated in 20 mL of TSB and BHI broth at 37 ◦ C with mild shaking at 150 rpm for 16 h. The grown culture was centrifuged at 3000 rpm, and the pellet was suspended in sterile TSB and BHI broth, followed by 10 times dilution with sterile distilled water. Film samples (about 100 mg) were incubated at 37 ◦ C for 15 h under mild shaking in 50 mL of diluted broth (106 –107 CFU/mL bacteria). The sampling was done at every 3 h, and the cell viability of each pathogen was determined by counting bacterial colonies on the plates. 2.7. Statistical analysis Statistical analysis was done using SPSS for Windows (SPSS Inc., Chicago, IL, USA) to test analysis of variance (ANOVA). Duncan’s multiple range tests were performed to determine the significant difference between treatments at a 95% confidence level. 3. Results and discussion 3.1. Properties of silver nanoparticles The AgNPs were prepared by reducing AgNO3 with aqueous CMLE. Initially, the mixed solution of CMLE and AgNO3 was transparent with a yellowish tint, and it changed to bright yellow as AgNO3 was reduced. The color development in the solution indicated the formation of AgNPs, in which the Ag ions (Ag+ ) were reduced to metallic AgNPs (Ag0 ). On the contrary, the CMLE and AgNO3 used as the control did not show any change in color. During the process, samples were taken at a different time interval and determined UV–vis spectra in the range of 200–700 nm and the results were shown in Fig. 1. The solutions exhibited absorp-

WVP = (WVTR × L)/p Where L was the thickness of the film (m) and p was partial water vapor pressure difference (Pa) across the film. To determine p, the actual water vapor pressure underneath the film sample was calculated by the method of Gennadios et al. [15]. This experiment was performed in triplicate with individually prepared films. Water contact angle was measured using a WCA analyzer (model Phenix 150, Surface Electro Optics Co., Ltd., Kunpo, Korea) after ca. 10 ␮L water drops placed on the surface of films using a microsyringe. This experiment was performed in triplicate with individually prepared films. For obtaining moisture content (MC) of the films, each film was cut into 3 cm × 3 cm and dried at 100 ◦ C for 24 h using an oven. The weight loss of each film was measured as MC and expressed as percent MC based on the initial weight of the film. This experiment was performed in triplicate with individually prepared films. 2.5.5. Thermal stability Thermal stability of the films was evaluated using a thermogravimetric analyzer (TGA; Hi-Res TGA 2950, TA Instrument, New

Fig. 1. UV–vis spectra of AgNPs at different time interval. The figure inset shows the TEM micrograph of AgNPs.

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Fig. 2. SEM micrographs of the surface and cross-section of the pectin-based composite films.

tion peaks at 420–502 nm depending on the process time, which was due to the surface plasmonic effect of AgNPs. The intensity of the peaks increased with an increase in reduction time and they showed the red-shift with the progress of time. The broadening and red-shift of peaks might be due to the growth of the size of AgNPs with time [18]. The broadening and red-shift of the peak are known to depend on the morphology of nanoparticles as well as on the types of reducing and stabilizing agents [19]. The shift of peak to higher wavelength with time was also reported when Rhodomyrtus tomentosa acetone extract was used as reducing and stabilizing agents for the synthesis of AgNPs [10]. The peaks between 230 and 350 nm might be due to the presence of phenolic compounds in the CMLE. A typical TEM micrograph of AgNPs was also shown in Fig. 1. The TEM image showed that the AgNPs formed were spherical in shape with the size of 20–80 nm. It also shows the AgNPs were separated individually, without distinctive agglomeration. This is presumably due to the stabilization of the AgNPs by the CMLE, which is characteristic of well-dispersed AgNPs. 3.2. Properties of pectin/AgNPs nanocomposite films 3.2.1. Morphology, surface color, and optical properties All the films (neat pectin, pectin/CMLE, and pectin/AgNPs) were flexible and freestanding. The surface morphology and crosssection of each film observed using the FE-SEM micrographs was shown in Fig. 2. The neat pectin and pectin/CMLE films exhibited a smooth and compact surface, while pectin/AgNPs nanocompos-

ite films showed rough surface structures with evenly distributed AgNPs. The AgNPs were homogeneously distributed on the surface through the whole film as observed in both surface and crosssectional view. Apparently, the neat pectin and pectin/CMLE films were translucent with a whitish tint. However, the pectin/AgNPs film was brownish yellow in color (Fig. S1, Supplementary data). The surface color of films determined by the Hunter L, a, b-values was shown in Table 1. The lightness (Hunter L-value) of pectin film was 89.90, but it decreased significantly (p < 0.05) to 88.60 and 40.77 after blending with CMLE and AgNPs, respectively. Hunter a- and b-values (indicating greenness-redness and blueness-yellowness, respectively) of pectin/CMLE film were not significantly different from those of the neat pectin film. The L-value of AgNPs-incorporated pectin films decreased, whereas, Hunter a- and b-values of pectin/AgNPs composite film increased significantly (p < 0.05). The change in the color values of the pectin/AgNPs composite film was mainly attributed to the AgNPs with brown color. Consequently, the total color difference (E) of pectin/AgNPs nanocomposite films (56.46) increased remarkably compared with the neat pectin film (7.52). This result is in good accord with the visible observation. Similar results were observed with other biopolymer films such as gelatin and agar-based films incorporated with AgNPs [6,20]. The optical properties of the neat pectin, pectin/CMLE, and pectin/AgNPs films were determined by measuring the absorption in the range of 200–700 nm and the percent transmittance at 280 and 660 nm. The UV–vis absorption spectra of pectin-based films were also shown in Fig. 3. The neat pectin and pectin/CMLE

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Table 1 Surface color and transmittance of the pectin-based filmsa . Film

L

a

b

E

T660 (%)

T280 (%)

Pectin Pectin/CMLE Pectin/AgNPs

89.90 ± 0.39a 88.60 ± 0.83b 40.77 ± 0.74c

−0.02 ± 0.04b 0.28 ± 0.11b 14.16 ± 1.09a

8.80 ± 0.59b 8.19 ± 0.81b 11.90 ± 0.62a

7.52 ± 0.70b 8.03 ± 1.13b 56.46 ± 0.41a

89.5 ± 0.6a 88.6 ± 1.5a 56.5 ± 1.4b

30.1 ± 4.3a 10.4 ± 1.7b 2.9 ± 0.7c

a Each Values are the mean of three replicates with the standard deviation. Any values in the same column followed by the same letter are not significantly (p > 0.05) different by Duncan’s multiple range tests.

Fig. 3. UV–vis spectra pectin-based composite films.

Fig. 4. FTIR spectra of pectin-based composite films.

films showed the absorption peaks in the ultraviolet range (below 300 nm) without specific absorption in the visible range. However, pectin/AgNPs composite film exhibited a profound absorption peak at 480 nm, which was attributed to the AgNPs [6]. The characteristic peak of AgNPs of the pectin/AgNPs composite film showed a blue shift (shift from 502 nm to 480 nm) after forming the composite with pectin polymer (see Fig. 1). In contrast, the red shift of AgNPs peak is reported in agar/AgNPs composite film [6]. This indicates that the direction of the shift of AgNPs peak probably depends on the type of polymer and compatibility between AgNPs and the polymer. Table 1 also showed the percent light transmittance of the pectin-based films determined at 660 and 280 nm, which indicates transparency and UV-screening properties of the films, respectively. The T660 of the pectin and pectin/CMLE films were 89.5 and 88.6%, respectively, which indicated they were highly transparent. However, T660 of the pectin/AgNPs composite film decreased significantly (p < 0.05). The T280 of the neat pectin film was 30.1%, which indicated that the pectin film is a fairly good UV barrier since it prevented 70% of incident UV light. However, UV-screening function of pectin film was further increased after addition of CMLE and AgNPs, in which about 90% and 97% of UV-light was screened in the pectin/CMLE and pectin/AgNPs film, respectively. The increase of UV barrier property of those composite films was presumably due to phenolic compounds in the CMLE and AgNPs [11].

3.2.2. FT-IR analysis The FT-IR spectra of neat pectin, pectin/CMLE, and pectin/AgNPs nanocomposite films exhibited distinctive peaks in the range of 4000–600 cm−1 (Fig. 4). A broad peak at 3330 cm−1 was due to the stretching vibrations of O H, and bands at 2930–2850 cm−1 were attributed to the C H stretching vibrations of methylene groups and the methyl group of pectin polymer chains [21]. The peaks at 1624 and 1440 cm−1 were assigned to asymmetric and symmetric stretching vibrations of carboxyl groups present in the pectin. The peaks of 1737 and 1228 cm−1 were attributed to the C O and C O component of an ester bond, respectively [22]. The bands at 1100 and 1020 cm−1 were assigned to C O C stretching vibrations of the saccharide structure. Similar peaks were observed in pectin/CMLE and pectin/AgNPs composite films, which suggested that the chemical structure of pectin was not changed after incorporation of AgNPs. However, the intensity of the peak at 3330 cm−1 increased after composite formation with CMLE and AgNPs. This result suggested that the CMLE and AgNPs interacted with pectin by the hydrogen bond. 3.2.3. Mechanical properties Table 2 shows the mechanical properties of the pectin-based films. The thickness of the neat pectin film was 40.5 ␮m, which was not changed significantly (p < 0.05) after blending with CMLE or

Table 2 Mechanical properties, MC, WVP, and WCA of the pectin-based filmsa . Film

Thickness (␮m)

TS (MPa)

Pectin Pectin/CMLE Pectin/AgNPs

40.5 ± 3.5 40.5 ± 4.7a 39.9 ± 2.2a

23.3 ± 4.4 22.9 ± 3.6a 25.2 ± 3.3a

a

EAB (%) a

E (MPa)

21.6 ± 6.9 20.6 ± 7.0a 19.0 ± 3.6a

a

MC (%)

472.9 ± 121.0 456.4 ± 97.8a 513.6 ± 48.1a

a

9.4 ± 0.5 10.2 ± 0.6b 8.6 ± 0.4a ab

WVP (×10−9 g m/m2 Pa s)

WCA (deg)

0.99 ± 0.04 0.91 ± 0.18a 0.91 ± 0.13a

51.1 ± 3.9a 51.4 ± 2.8a 49.5 ± 2.8a

a

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

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AgNPs. Though the mechanical properties (TS, EAB, and E) of pectin film changed slightly after blending with CMLE or AgNPs, they were not significantly different (p > 0.05) from those of neat pectin film. The TS of the pectin film increased from 23.3 MPa of the pristine pectin film to 25.2 MPa of the pectin/AgNPs composite film. Similarly, the E of pectin film increased from 472.9 MPa to 513.6 MPa after incorporation of AgNPs. In contrast, the EAB decreased from 21.6% to 19.0% after incorporation of AgNPs. The slight increase in mechanical strength of the pectin/AgNPs composite film might be due to the interaction formed by the hydrogen bond between AgNPs and pectin polymer as seen in the results of FT-IR. The mechanical properties of the films are closely related to the distribution and density of the intra and intermolecular interactions between the polymer chains in the film matrix [23]. 3.2.4. Moisture content, water vapor permeability, and water contact angle The WVP, WCA, and MC of the pectin-based films were presented in Table 2. The MC of the neat pectin film was 9.4%. The MC of the pectin blend films was influenced by the type of the fillers, i.e., the MC of pectin/CMLE films increased to 10.2%, while that of pectin/AgNPs films decreased down to 8.6%. This was presumably due to that the CMLE was more hydrophilic and the AgNPs were more hydrophobic than the polymer matrix. The WVP of the neat pectin film was 0.99 × 10−9 g m/m2 Pa s which decreased to 0.91 × 10−9 g m/m2 Pa s after blending with both CMLE and AgNPs, however, the change was not statistically significant (p > 0.05). Surface hydrophobicity of the pectin, pectin/CMLE, and pectin/AgNPs composite films was tested by measuring the contact angle of water on the surface of the films, and the results were also shown in Table 2. The WCA of the neat pectin film was 51.1◦ , which decreased slightly to 49.5◦ after the formation of a composite film with AgNPs. The decrease in the WCA of the pectin/AgNPs films might be due to the increase in roughness of composite films after incorporation of metallic AgNPs. The WCA is a characteristic property of a film, which is usually used as a measure of wettability of the films. Usually, the film surface is considered hydrophilic when the WCA is lower than 65◦ [24]. Since the film matrix (pectin) is hydrophilic in nature, the resulting film formed a hydrophilic surface with low WCA values. 3.2.5. Thermal stability The thermal stability of the pectin, pectin/CMLE, and pectin/AgNPs films was tested using TGA, and the resulting TGA and DTGA curves were presented in Fig. 5. The TGA curves of the films exhibited the weight loss pattern and the DTGA curves clearly showed the maximum decomposition temperature (Tmax ) at each degradation step during thermal decomposition. All the films exhibited three distinctive steps of thermal degradation. The first step of thermal degradation occurred at around 50 ◦ C corresponding to the evaporation of moisture, which was about 5–7% of initial weight [25]. The second and main step of thermal degradation was observed between 170–240 ◦ C, which corresponded to the degradation of glycerol and biopolymer pectin. The third step of thermal degradation was observed around 320–350 ◦ C which was mainly due to the oxidation of polymer and CMLE [26]. After the final thermal decomposition, the residuals of the neat pectin, pectin/CMLE, and pectin/AgNPs films were 16.1, 27.6, and 26.2%, respectively (Table 3). The higher residuals of pectin/CMLE and pectin/AgNPs films might be due to the presence of CMLE and AgNPs in the composite films, respectively. 3.3. Antibacterial activity The antibacterial activity of synthesized AgNPs (using CMLE) in terms of minimum inhibitory concentrations (MIC) and minimum

Fig. 5. (a) TGA and (b) DTGA spectra of pectin-based composite films.

Table 3 Results of thermogravimetric analysis of composite films. Film

Tmax (◦ C)

Decomposition (%)

Char content at 600 ◦ C (%)

Pectin

47.5 187.5 222.5

3.51 23.85 40.53

16.1

Pectin/CMLE

47.5 187.5 222.5

2.01 18.06 32.33

27.6

47.5 187.5 222.5

1.72 18.22 32.95

26.2

Pectin/AgNPs

Tmax is the maximum decomposition temperature at each decomposition step.

bactericidal concentrations (MBC) against E. coli and L. monocytogenes were 8/16 and 16/64 ␮g/mL, respectively. However, CMLE exhibited MIC/MBC of >1000/>1000 ␮g/mL against both E. coli and L. monocytogenes. AgNPs are considered as antimicrobial agents with a broad spectrum of bacterial and fungal species [27]. Owing to the nanosize with very high surface area, AgNPs shows very strong antimicrobial activities [10,28]. However, AgNPs are known to have lower toxicity towards mammalian cells than bacteria when biological molecules are used as reducing and capping agents [29]. The antimicrobial activity of pectin-based composite films was also tested against L. monocytogenes and E. coli using viable

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to possess antibacterial activity by either rupturing the negatively charged bacterial cell wall or by destabilizing the outer membrane, thereby providing them access to the mitochondria and resulting in the interference with the respiratory chain [30,31]. Even though the use of AgNPs in biomedical applications is well documented, their utilization in the food packaging area is limited. The pectinbased AgNPs composite films developed in the present study are expected to be utilized for the food packaging applications in the form of packaging film and edible coating to prolong the shelf-life of packaged foods. 4. Conclusions The AgNPs were synthesized using CMLE as reducing and capping agents, and they were used to prepare pectin based antimicrobial nanocomposite films. The AgNPs was 20–80 nm in size and exhibited maximum absorption peak at 502 nm. However, pectin/AgNPs composite films exhibited blue shift with a maximum absorption peak at around 480 nm. The mechanical, water vapor barrier, and thermal properties of the films have increased slightly, however, optical properties have enhanced significantly by the incorporation of AgNPs. The All pectin based composite films showed UV barrier property, and this was enhanced further with the incorporation of AgNPs. In addition, the pectin/AgNPs composite films had stronger antimicrobial activity against E. coli than L. monocytogenes. The results suggest that the prepared pectin/AgNPs composite films with improved properties and antimicrobial activity have a high potential for the use as an active food packaging to increase the food safety and prolong the shelf-life of packaged foods. Acknowledgements This research was supported by the Agriculture Research Center (ARC 710003) program of the Ministry of Agriculture, Food, and Rural Affairs, Korea. Appendix A. Supplementary data Fig. 6. Antimicrobial activity of pectin-based composite films against (a) L. monocytogenes and (b) E. coli.

colony count methods, and the results were presented in Fig. 6. The neat pectin and pectin/CMLE films did not show any antimicrobial activity against both bacteria. However, the AgNPs incorporated pectin composite films demonstrated stronger antimicrobial activity against E. coli than L. monocytogenes. The disc diffusion method was also performed to check the antibacterial activity of pectinbased composite films. The film disc of 10 mm diameter was placed on the agar plated pre-inoculated with bacteria and incubated the plate at 37 ◦ C for 20 h and the clear zone around films were measured. The zone of inhibition in mm was determined by subtracting the diameter of the film disc from the total diameter of inhibited zone. As expected, the neat pectin and pectin/CMLE composite films did not show any zone of inhibition. However, pectin/AgNPs composite film exhibited clear zone of inhibition against both, E. coli (8.4 ± 1.2 mm) and L. monocytogenes (3.9 ± 0.8 mm). The difference in the antibacterial activity of AgNPs between Gram-positive and Gram-negative bacteria might be due to the difference in the structure of their cell wall. Although the clear mechanism of antibacterial activity of AgNPs has not been clearly known, it has been believed that positively charged silver ions interact with negatively charged biomolecules (DNA and protein) causing structural changes that lead to disruption of metabolic processes followed by cell death. The Ag+ cations and partially oxidized AgNPs have been shown

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