Accepted Manuscript Title: Antimicrobial nanostructured starch based films for packaging Author: Ana S. Abreu M. Oliveira Rui M. Rodrigues Miguel A. Cerqueira Ant´onio A. Vicente A.V. Machado PII: DOI: Reference:
S0144-8617(15)00330-6 http://dx.doi.org/doi:10.1016/j.carbpol.2015.04.021 CARP 9852
To appear in: Received date: Revised date: Accepted date:
9-12-2014 5-3-2015 8-4-2015
Please cite this article as: Abreu, A. S., Oliveira, M., Rodrigues, R. M., Cerqueira, M. A., Vicente, A. A., and Machado, A. V.,Antimicrobial nanostructured starch based films for packaging, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.04.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antimicrobial nanostructured starch based films for packaging
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Ana S. Abreu a,*, M. Oliveira a, Rui M. Rodrigues b, Miguel A. Cerqueira b, António A.
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Vicente b and A. V. Machado a a
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Institute of Polymers and Composites (IPC) and Institute of Nanostructures,
Nanomodelling and Nanofabrication (I3N), University of Minho, Campus de Azurém,
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4800-058 Guimarães, Portugal b
Centre of Biological Engineering (CEB), University of Minho, Campus de Gualtar,
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4710-057 Braga, Portugal
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Corresponding Author: Ana Sofia Abreu
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Institute of Polymers and Composites (IPC)/ Institute of Nanostructures Nanomodelling
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and Nanofabrication (I3N)
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Campus de Azurém,
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4800-058 Guimarães, Portugal
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Phone: +351 253 510320;
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Fax: +351 253 510339
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[email protected] 19
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ABSTRACT
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Montmorillonite modified with a quaternary ammonium salt C30B/starch nanocomposite
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(C30B/ST-NC), silver nanoparticles/starch nanocomposite (Ag-NPs/ST-NC) and both
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silver nanoparticles/C30B/starch nanocomposites (Ag-NPs/C30B/ST-NC) films were 1 Page 1 of 29
produced. The nanoclay (C30B) was dispersed in a starch solution using an ultrasonic
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probe. Different concentrations of Ag-NPs (0.3, 0.5, 0.8 and 1.0 mM) were synthesized
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directly in starch and in clay/starch solutions via chemical reduction method. Dispersion of
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C30B silicate layers and Ag-NPs in ST films characterized by X-ray and scanning electron
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microscopy showed that the presence of Ag-NPs enhanced clay dispersion. Color and
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opacity measurements, barrier properties (water vapor and oxygen permeabilities), dynamic
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mechanical analysis and contact angle were evaluated and related with the incorporation of
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C30B and Ag-NPs. Films presented antimicrobial activity against Staphylococcus aureus,
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Escherichia Coli and Candida Albicans without significant differences between Ag-NPs
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concentrations. The migration of components from the nanostructured starch films,
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assessed by food contact tests, was minor and under the legal limits. These results indicated
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that the starch films incorporated with C30B and Ag-NPs have potential to be used as
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packaging nanostructured material.
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Starch;
antimicrobial
activity;
silver
nanoparticles;
organoclay;
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KEYWORDS:
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nanostructured films; food packaging.
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1. Introduction
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The development of nanostructured materials, revealed a promising advance in
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nanotechnology, materials and life sciences (Ojijo & Sinha Ray, 2013). They can be
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composed by a natural polymer matrix and organic/inorganic reinforcement with at least
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one dimension on the nanometer scale (Darder, Aranda & Ruiz-Hitzky, 2007). Moreover,
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these materials show the extraordinary advantages of biocompatibility and biodegradability 2 Page 2 of 29
in medical, drug release, packaging and agricultural applications (Mangiacapra, Gorrasi,
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Sorrentino & Vittoria, 2006; Reddy, Vivekanandhan, Misra, Bhatia & Mohanty, 2013;
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Sorrentino, Gorrasi & Vittoria, 2007; Sozer & Kokini, 2009).
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Among natural polymers, starch is one of the most promising biocompatible and
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biodegradable material, which has received attention due to its strong advantages, such as,
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low cost, wide availability from many plants and total compostability without the formation
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of toxic residues (Mathew & Dufresne, 2002). In their native form, starches are organized
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into semicrystalline granules, presenting poor mechanical properties and high water
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affinity. The addition of nanofillers aims to improve some of these properties. In contrast,
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the macromolecular chains of this biopolymer possess a large number of hydroxyl groups,
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which is excellent for metal complexation. The hydroxyl groups provide active sites for
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metal ions enabling a good control of size, shape and dispersion of the formed metallic
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nanoparticles (Raveendran, Fu & Wallen, 2006). The development of nanostructured
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materials with metallic nanoparticles, such as silver nanoparticles (Ag-NPs), is highly
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useful to minimize the growth of contaminants by microorganisms. Therefore, there is a
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raising interest in developing bio-based polymers with antimicrobial activity. Silver
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nanoparticles (Ag-NPs) are known to have inhibitory and antimicrobial properties and low
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toxicity, receiving special attention by the industry. Ag-NPs can also be employed as
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absorbent pads in food packaging to absorb moisture and fluids exuded from meat and fish,
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keeping the products looking fresh and creating an aesthetically attractive packaging (Cho,
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Park, Osaka & Park, 2005; Duncan, 2011). The Ag-NPs interactions with microorganisms
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are dependent on size and shape of the nanoparticles but the inhibition mechanism of Ag-
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NPs remains unclear (Pal, Tak & Song, 2007).
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The simplest and most direct route to metal nanoparticles synthesis is in situ generation
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inside a solid polymer film, which involves the preparation of thin polymeric film
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containing the metal precursor, that will generate the nanoparticles within the polymer
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matrix through a variety of chemical and physical methods (Ramesh, Porel &
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Radhakrishnan, 2009).
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Nanoclay have been largely used to improve bio-nanocomposite mechanical properties and
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also demonstrated antimicrobial activity. Rhim et al. studied antimicrobial activity of
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chitosan/clay films prepared with two different types of nanoclays (i.e. a natural
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montmorillonite - MMT and an organically modified montmorillonite - Cloisite 30B). It
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was shown that the materials prepared with the Cloisite 30B exhibited antibacterial activity
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against Gram-positive bacteria, but the natural MMT did not show any antibacterial activity
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(Rhim, Hong, Park & Ng, 2006).
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Nanostructured starch materials have been mainly prepared by solution casting
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(Majdzadeh-Ardakani, Navarchian & Sadeghi, 2010), coprecipitation (Chung, Ansari,
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Estevez, Hayrapetyan, Giannelis & Lai, 2010) and melt intercalation (Lee, Chen & Hanna,
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2008). Starch films with Ag-NPs (7.8 mM) prepared by solution casting, where Ag-NPs
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were reduced using sodium borohydride proved to prevent the viability and growth of
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pathogens such as: Staphylococcus aureus, Escherichia coli and Candida albicans
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(Božanić et al., 2011).
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According to literature, the incorporation of both organoclay and silver nanoparticles into
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polymer matrices, to achieve improved mechanical and barrier properties and antimicrobial
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activity was not yet studied. Therefore, the present work aims to develop a nanostructured
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starch based film containing C30B clay (3 wt. %) and silver nanoparticles with enhanced
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mechanical, barrier and antimicrobial properties. Nanocomposite films of clay/starch,
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silver/starch and silver/clay/starch (C30B/ST-NC, Ag-NPs/ST-NC and Ag-NPs/C30B/ST-
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NC) were prepared by solution casting method. The silver nanoparticles (Ag-NPs) were
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incorporated and synthesized in-situ by chemical reduction method. Clay dispersion in
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starch films was characterized by X-ray diffraction (XRD), scanning electron microscopy
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(SEM) and influence on mechanical properties assessed by dynamical mechanical analysis
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(Lan, Kaviratna & Pinnavaia). As critical parameters for some applications, like food
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packaging, contact angle, color and opacity, permeability to water vapor (PWvapor) and
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oxygen (PO2) and contact tests were performed. Antimicrobial activity of the prepared
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nanostructured films was determined in order to evaluate the effect of different Ag-NPs
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concentrations.
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2. Experimental
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2.1. Materials
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Native corn starch (ST) was kindly supplied by PIEP - Innovation in Polymer Engineering
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(Portugal). Silver nitrate (AgNO3) and sodium borohydrate (NaBH4) in powder state were
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used as received and supplied by Sigma Aldrich (St. Louis, MO, USA) and Acros Organics
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(Geel, Belgium), respectively. Cloisite® 30B (C30B) a montmorillonite modified with a
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quaternary ammonium salt (MT2EtOH = methyl tallowyl bis-2-hydroxyethyl ammonium
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chloride), was used as received and supplied by Southern Clay Products, Inc. (Texas,
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USA).
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2.2. Film preparation
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2.2.1. C30B dispersion in ST solution (C30B/ST)
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An aqueous starch solution (10 g/L) was heated at 70-80 ºC until the complete dissolution
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of starch. Then, 3 wt. % of C30B was added and the mixture was sonicated using an
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ultrasonic probe, at room temperature, for 1 h. Percentage amplitude of 100 % as power
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delivery was used. An ultrasonic processor UP100H with a MS7D flow tip 7 (Teltow,
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Germany) was used with a 30 kHz frequency.
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2.2.2. Ag-NPs/C30B/ST-NC films preparation
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Ag-NPs/C30B/ST-NC films were prepared as follows: 25 mL of the C30B/ST dispersed
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solution (please see 2.2.1) and different concentration of silver nitrate (0.3, 0.5, 0.8 and 1.0
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mM) were mixed and left stirring during 5 min. Then, the reducing agent, sodium
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borohydride, was slowly added at the same concentrations as silver nitrate. The color of the
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prepared solutions with different concentration of AgNO3 changed from colorless to light
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brown then to brown and finally to dark brown, indicating the formation of Ag-NPs in
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C30B/ST solution. All the mixtures were boiled during 5 min, including ST and C30B/ST
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solution as controls. Ag-NPs/ST-NC nanostructured films were prepared using the same
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procedure without the addition of C30B to ST solution.
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Different concentrations of silver/starch (Ag-NPs/ST-NC), silver/clay/starch (Ag-
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NPs/C30B/ST-NC) solutions (25 mL each) were spilled in glass plates (7.0 x 1.5 cm). All
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solutions were dried at room temperature for 48 h and then placed in an oven at 25 °C in
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vacuum for one additional day (Figure 1). The mean diameter of the prepared films was
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adjusted to the same size. All films were conditioned at 50 % relative humidity and room
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temperature before analysis. The same procedure was applied to starch and to clay/starch
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(C30B/ST-NC) solutions as blanks (Figure 1).
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Figure 1. Nanostructured starch films.
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2.3. Characterization
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2.3.1. XRD
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X-ray diffraction measurements (XRD) were performed to evaluate the dispersion of C30B
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in the starch films. XRD spectra of the samples were obtained at room temperature using a
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diffractometer (AXS Nanostar- D8 Discover, Bruker) equipped with a CuK generator ( =
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1.5406 Å) at 40 mA and 40 kV. Data were collected in a range of 0.08 to 10 with a step
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size of 0.01º and a counting time of 2 seconds per step. The clay powder or starch
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nanocomposite films were analyzed directly. The Bragg´s law (
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to determine the clay interlayer distance d001, where d is the spacing between silica layers of
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the clay (also called interlayer spacing), the wavelength of X-ray on the silica layer, and n
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is a whole number which represents the order of diffraction, taken 1 in our calculations.
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2.3.2. SEM
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Scanning electron microscopy (SEM) was carried out to examine the surface morphology
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of starch nanocomposites. The SEM analysis was performed in a Leica Cambridge S360
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microscope in back-scatter mode. The samples were previously fractured in liquid nitrogen
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and coated with a gold thin film.
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2.3.3. DMA
was used
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Dynamic mechanical analyses (DMA) (Lan, Kaviratna & Pinnavaia) experiments were
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performed in a TRITON apparatus in tension mode with the following parameters:
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frequency = 1 Hz; scan rate = 2 ºC/min; temperature range = -70 to 100 ºC; free length = 15
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mm and value tension = 15 m. The equipment was calibrated according to the standard
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procedure. The tests were performed on 3 x 30 mm rectangular samples in a longitudinal
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direction. At least two specimens of each sample were tested.
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2.3.4. Color and opacity
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Color and opacity of the films were evaluated using a Konica Minolta, Chroma Meter (CR
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410; Konica Minolta, Japan). The CIELab scale was used to determined L*, a* and b*
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color parameters. L* is the vertical coordinate of a three-dimensional system of colors,
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which has values from 0 (black) to 100 (for white), a* is the horizontal coordinate the
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values of which range from –80 (green) to +80 (red) and b* is the horizontal coordinate the
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values of which range from –80 (blue) to +80 (yellow). The opacity (Y) was determined
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according to the Hunterlab method (Hunterlab, 2008), as the ratio between the opacity of
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each sample on the black standard (Yb) and the opacity of each sample on the white
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standard (Yw). The results were expressed as a percentage: Y (%) = 100 (Yb/Yw). From three
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replicates of each film sample were determined at random three measurements, L*, a*, b*,
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Yb and YW and an average of each one was used for calculation.
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2.3.5. Contact angle
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The influence of chemical modification on the surface polarity of starch films was studied
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by means of contact angle measurements. The Young-Laplace method for the water drop
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was used to determine the contact angle (Krishnan, Liu, Cha, Woodward, Allara & Vogler,
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2005). One drop of 3 L was placed on the surface of the films and a Contact Angle System
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OCA 15 Plus with CCD video camera (resolution of 752 x 582 pixel) and C20 software
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was used. The contact angle was calculated from the shape of the drop according to the
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following equation: = 2 tan -1(2 h/d), where h and d are the height and diameter of the
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water drop, respectively. Four measurements were performed for each material.
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2.3.6. Permeability to Gases
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Water vapor permeability (PWvapor) was determined by gravimetric method based on
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Casariego et al. procedure (Casariego et al., 2009). Film samples were placed on top of
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cups containing 50 mL of distilled water and properly sealed (films were sealed by the
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edges using a sealant ring), being the initial weight determined. Afterwards samples were
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placed inside a desiccator containing silica gel (0 % RH; 20 °C), using a fan to guarantee
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the homogenization of the gases inside the desiccator (Duncan, 2011). To monitor weight
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loss over time, periodical cup weightings (2 h) were performed until steady state was
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reached. Water vapor transmission rate (WVTR) was calculated by dividing the slope of the
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linear regression of weight loss by film area, and PWvapor (g/m.s.Pa) was calculated as
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follows:
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where L is film thickness (m) and Δp is water vapor partial pressure difference (Pa) across
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the two sides of the film.
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Oxygen permeability (PO2) measurements were conducted based on Cerqueira et al.
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method (Cerqueira, Souza, Teixeira & Vicente, 2012). The tests were performed at 20 °C in
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a gas permeation cell composed by two chambers with test film placed between them. The
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permeation gas, oxygen, flowed continuously through the lower chamber, and nitrogen (as
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a sweep gas) was passed through the upper chamber. When the steady state was reached,
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the penetrated O2 in the sweep gas stream was analyzed by gas chromatography
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(Chrompack 9001, Middelburg, Netherlands) at 110 °C with a column Porapak Q 80/100
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mesh 2 m × 1/8 in. × 2 mm SS equipped with a hydrogen flame ion detector. Helium was
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used as carrier gas at 23 mL/min. A standard mixture containing 10 % of CO2, 20 % of O2
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and 70 % of N2 was used for calibration.
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For all determinations, at least three repetitions were made for each type of film.
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2.4. Statistical Analysis
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Statistical analysis was performed using the analysis of variance (Mulinacci et al.)
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procedure with SigmaPlot 11.0 software for Windows. Tukey’s test was applied to detect
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differences of means, and p0.05) in color and opacity values. Still, the
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incorporation of Ag-NPs changes (p