Accepted Manuscript Title: Microstructure and physical properties of nano-biocomposite films based on cassava starch and laponite Authors: Germ´an Ayala Valencia, Carla Giovana Luciano, Rodrigo Vinicius Lourenc¸o, Paulo Jos´e do Amaral Sobral PII: DOI: Reference:
S0141-8130(17)33048-9 https://doi.org/10.1016/j.ijbiomac.2017.10.031 BIOMAC 8326
To appear in:
International Journal of Biological Macromolecules
Received date: Accepted date:
15-8-2017 5-10-2017
Please cite this article as: Germ´an Ayala Valencia, Carla Giovana Luciano, Rodrigo Vinicius Lourenc¸o, Paulo Jos´e do Amaral Sobral, Microstructure and physical properties of nano-biocomposite films based on cassava starch and laponite, International Journal of Biological Macromolecules https://doi.org/10.1016/j.ijbiomac.2017.10.031 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.
Microstructure and physical properties of nano-biocomposite films based on cassava starch and laponite Germán Ayala Valencia*, Carla Giovana Luciano, Rodrigo Vinicius Lourenço, Paulo José do Amaral Sobral**. Department of Food Engineering, Faculty of Animal Science and Food Engineering, University of São Paulo, Pirassununga, SP, Brazil. *Corresponding *[email protected]; **[email protected] Abstract The aim of this research was to study the effects of laponite concentrations on some properties of nano-biocomposite films based on cassava starch, focusing mainly the relation between the properties of the surface microstructure and roughness, water contact angle and gloss. Nano-biocomposite films were produced by casting. We analyzed gloss, color, opacity, water contact angle, crystallinity by X-ray diffraction, and microstructure by scanning electron microscopy and atomic force microscopy. Texture parameters (energy, entropy and fractal dimension) were extracted from micrographs. We observed a great impact of laponite in the morphology of nanobiocomposite films. Texture parameters correlated with surface heterogeneity and roughness. Finally, surface roughness affected the surface hydrophilicity of nanobiocomposite films. Laponite platelets were exfoliated and/or intercalated with amylose and amylopectin chains. This research reports new information on the effects of laponite concentrations on the morphological, optical and wetting properties of nano-biocomposite films aiming future industrial applications.
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Keywords: gloss; image analysis; nano-biocomposite films; microstructure; water contact angle. 1. Introduction The development of nanostructured materials has a promising advance in several areas such as food packaging. Normally, such nanostructured materials are composed by a natural polymer matrix, called biopolymer, and an organic/inorganic particle loading, which are characterized by having at least one dimension between 1 and 100 nm [1, 2]. Starch is a good candidate for the biopolymer matrix of nanostructured materials because it is cheap, abundant, renewable, biocompatible and biodegradable [1], however, films based on starch present poor mechanical properties and a high water sensibility [3, 4]. Several methods have been studied to avoid or reduce this behavior such as the use of different plasticizers [4, 5], blending starch with a biodegradable synthetic polymer such as poly(vinyl alcohol) [6], or even the use of flours [7], which are natural blends of starch with proteins [8]. All cases resulted in a relative success regarding the improvement of the overall properties of films. A recent alternative to improve the physical properties of starch films is a reinforcement with nanoclays [1, 9-12], especially montmorillonite (Mt), which has been the nanoclay most studied for starch films [1, 9-11, 13]. Another nanoclay not commonly studied for starch-based films is laponite (Lap) [2, 9, 11]. Lap (hydrous sodium lithium magnesium silicate) is a crystalline layer silicate with a structure and a composition that closely resembles smectite (Sm) [14]. Its chemical formula is Si 8[Mg5.5Li0.4H4.0O24.0]0.7-[Na0.7]0.7+ [15]. The Lap nanoparticle has
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the shape of a disk with a thickness of approximately 1 nm and a diameter of approximately 25 nm, being smaller than raw clays such as Mt [14]. Lap has been successfully dispersed into cellulose and gelatin matrixes, improving the mechanical and water vapor permeability properties without changing the optical properties of nano-biocomposite films [16-18]. Moreover, some starch nanobiocomposite films have been developed using the casting technique [2, 9, 11]. However, no study explored the relation between the surface microstructure and the surface properties of films affected by different Lap concentrations. Thus, the objective of this research was to study the effects of laponite concentrations on physical properties of nano-biocomposite films based on cassava starch, specially the relation between the surface microstructure and other surfaces (roughness, water contact angle and gloss) properties. 2. Materials and methods 2.1.
Materials
Cassava starch was purchased from Yoki Food Industry (São Paulo, Brazil) and was used as the biopolymer. Glycerol (Synth, Brazil) was used as a plasticizer, and laponite RD (Southerm Clay Products Inc. Reference number 23224) was used as the nanoparticle. The initial water content in cassava starch, glycerol and Lap was 12.4 ± 0.1, 14.1 ± 0.1 and 7.5 ± 0.1% (wet basis), respectively. Distilled water was used as the solvent. 2.2.
Preparation and characterization of nano-biocomposite films
Nano-biocomposite films were produced using a mixture of cassava starch (CS) (solution A) and laponite (Lap) dispersed into water containing glycerol (solution B). Solution A was prepared using 4 g of CS/100 g of solution, as follows: cassava starch 3
was heated at 90 ± 1°C for 30 min to gelatinize starch [19]. At the same time, solution B was prepared at room temperature using 1 g of Lap/100 g solution in distilled water and a high-speed homogenizer (ultraturrax, Ika, model T25) at 20,000 rpm for 30 min [19]. Then, glycerol (25 g of glycerol/100 g of CS) was added to the Lap dispersion and mixed for 15 min using a magnetic stirrer (Tecnal TE 0852, Brazil). Then, the solutions A and B were mixed conveniently to produce solutions with 0, 1.5, 3, 4.5 and 6 g of Lap/100 g of CS. After, the solutions were homogenized for 15 min at 90 ± 1°C using the same thermostatic water-bath. Nano-biocomposite films were produced by casting. The amount of solution poured into Petri dishes (diameter: 14 cm) was calculated to obtain a constant weight of dry matter of approximately 8 mg/cm2. All solutions were dried in a forced-air circulation oven (Marconi, MA037, Brazil) at 30°C and controlled relative humidity (RH = 60 ± 5%) for 20 hours. Nano-biocomposite films have a thickness of 80 ± 10 µm. For the SEM (scanning electron microscopy), AFM (atomic force microscopic) and X-ray diffraction analyses, samples were previously conditioned over silica gel, into desiccators for at least 7 days. For the characterization of gloss, color and contact angle, samples were conditioned into desiccators containing saturated solutions of Mg(NO3)2 (RH = 50.3%) at 25°C for at least 7 days. 2.2.1. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) The air and support surfaces of the nano-biocomposite films were fixed to aluminum stubs using a double-sided tape. The micrographs were taken at sample random positions with a 1000x magnification, using a scanning electron microscopy (SEM model TM-3000, Hitachi), at 5 kV [20]. The resolution of the micrographs was 0.13 4
µm/px and the field of view was 0.021 mm2. Five micrographs were made for each surface (air and support surfaces) for every nano-biocomposite film. The nano-biocomposite films were analyzed using an atomic force microscope (model NT-MDT Solver Next Brand, Russia), equipped with a software for image analysis. The nano-biocomposite films were analyzed using the semi-contact mode with a resonance frequency of 150 kHz, a contact force of 5 N/m and a scan speed 0.3 Hz [17]. Analyses were performed in 50 µm x 50 µm areas (field of view: 0.0025 mm2) using five different samples. The treatment of the images was performed using second-order subtraction and a software for images analysis (New Model 3.1.0 program PX). The average roughness (𝑅𝑎 ) and the root mean square roughness (𝑅𝑞 ) were calculated using the AFM software. All micrographs were made using RGB colors with a resolution of 0.2 µm/px. Texture image analysis was performed to quantify the surface microstructure captured by SEM and AFM micrographs. Three texture parameters (energy, entropy and fractal dimension) were analyzed using the software ImageJ v 1.39 (National Institute Health, Bethesda, MD, USA). Energy and entropy were determined in grayscale using the Gray Level Co-Ocurrence Matrix algorithm (GLCM) by fixing the distance of the pixels (d) to 1 and the angle of displacement (θ) to 0° [17, 21]. The fractal dimension (𝐹𝐷) was calculated based on gray-scale images using power-law scaling and Standard Box Count method [17, 21]. 𝐹𝐷 values were calculated taking into account the slope in the log(box count) vs. log(box size) plot, according to Arzate-Vázquez et al. [21]. 2.2.2. Gloss
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The gloss of nano-biocomposite films was determined using a gloss meter (Rhodopoint NGL 20/60), at angle of 60° in 10 points of each sample according to the ASTM D2457 standard [20]. Gloss measurements were performed on the air and the support surfaces of the nano-biocomposite films. 2.2.3. Films color and Opacity The color of nano-biocomposite films was determined using a Miniscan XE colorimeter (HunterLab), in the reflectance mode adopting the CIELab scale, an illuminant/angle D65/10° and an opening of 30 mm [17]. Samples were placed on the surface of the standard white plate and the parameters 𝐿∗ (lightness index), 𝑎∗ (color tonalities from red to green) and 𝑏∗ (color tonalities from yellow to blue) were determined. The total color difference (∆𝐸 ∗ ) was then calculated as: ∆𝐸 ∗ = √(∆𝐿∗ )2 + (∆𝑎∗ )2 + (∆𝑏∗ )2
(1)
∗ ∗ where∆𝐿∗ = 𝐿∗𝑠𝑎𝑚𝑝𝑙𝑒 - 𝐿∗𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 (92.58 ± 0.92); ∆𝑎∗ = 𝑎𝑠𝑎𝑚𝑝𝑙𝑒 - 𝑎𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 (-0.88 ± 0.06); ∗ ∗ ∆𝑏∗ = 𝑏𝑠𝑎𝑚𝑝𝑙𝑒 - 𝑏𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 (1.26 ± 0.02).
Opacity was measured using the same equipment of the color measurement, according to the Hunterlab method, in reflectance mode. Opacity (𝑌) values were 𝑌
calculated according the 𝑌 = 𝑌 𝑏 relation between the opacity of nano-biocomposite 𝑊
films superimposed over the black standard (𝑌𝑏 ) and the same sample superimposed over the white standard (𝑌𝑊 ). 2.2.4. Water contact angle The water contact angle of nano-biocomposite films was measured according to the ASTM D7334 standard and using an optical tensiometer (Attension Theta lite, KSV Instrument) [22]. The nano-biocomposite films were attached to the equipment and 6
one drop of distilled water of approximate 10 µL was dropped over the sample using an automatic precision syringe. The preparation was immediately photographed. The angle formed between the sample surface and the tangent of the drop was calculated using the software Attension Theta Lite. The measurements of water contact angle were made by analyzing the shape of one sessile drop after it had been placed over the samples for 30 seconds. The measurements of water contact angle were made on the air and the support surfaces of nano-biocomposite films. 2.2.5. X-ray diffraction X-ray diffractograms (XRD) of nano-biocomposite films were obtained using an Xray diffractometer (AXS Analytical X-ray Systems Siemens D 5005, Germany), operating at 40 kV and 40 mA (Cu Kα 1, λ = 1.54056 Å radiation). Samples (3 cm x 3 cm) were placed inside an aluminum frame. The spectra were recorded at 25°C between the angles 2θ = 2° and 50° at a rate of 2°/min [23]. Samples of Lap and cassava starch powders were also analyzed under the same conditions. The reflection peak, which is typical of Lap layers, was determined as 𝑑(001) applying the Bragg law (Equation 2) to the Lap spectrum and some films spectra whenever visible. 𝜆 = 2𝑑sinθ
(2)
where 𝜆 is the wavelength of Cu Kα radiation; 𝑑 is the spacing between Lap layers, and θ is the reflection angle. 2.3.
Statistical analyses
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Analysis of variance (ANOVA) and a Tukey test with multiple comparisons were performed with a significance level of 5% using the ‘‘Statistical Analysis Systems’’ software (version 9.2, SAS). 3. Results and discussions 3.1.
Films morphological properties
The support surfaces were smoother than the air surfaces of nano-biocomposite films (Fig. 1). Similar results were observed by Tang et al. [24] and Okeyoshi et al. [25], who studied films based on collagen and xanthan gum, respectively. The Lap concentration increased the air surface roughness of nano-biocomposite films. Big structures were observed in films containing 6 g of Lap/100 g of CS (see red arrows in Fig. 1f) probably due to the biopolymer aggregation. According to Cano et al. [26], nanoparticles could induce a phase separation among biopolymers of nanostructured films, promoting aggregations.
Fig. 1. AFM micrographs of films confirmed that the support surface roughness was lower than in the air surface (Fig. 2), as previously evidenced by the SEM micrographs (Fig. 1). Roughness values (𝑅𝑎 and 𝑅𝑞 ) confirmed the observations using SEM and AFM micrographs (Table 1): for control films (0 g of Lap/100 g of CS), 𝑅𝑎 and 𝑅𝑞 values in air surfaces were almost nine times higher than the 𝑅𝑎 and 𝑅𝑞 values recorded on the support surface. For control films, roughness values on the air surface were similar to those observed by Jiménez et al. [27], who studied corn starch films plasticized with glycerol.
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The roughness on the support surface decreased upon adding Lap from 12 to 6 nm (𝑅𝑎 ) and from 15 to 8 nm (𝑅𝑞 ), when the Lap concentration increased from 0 to 6 g of Lap/100 g of CS. Smoother surfaces added with Lap could be a consequence of biopolymer orientation on the support surface during the drying process. An opposite behavior was observed for the roughness on the air surface: 𝑅𝑎 increased from 112 to 161 nm and 𝑅𝑞 increased from 133 to 200 nm, when the Lap concentration increased from 0 to 6 g of Lap/100 g of CS. That is, the presence of Lap increased the roughness on the air surface of nano-biocomposite films probably due to the formation of agglomerates because of the interaction biopolymer chains–Lap platelets during the drying process of filmogenic solutions [20]. 𝑅𝑎 and 𝑅𝑞 values on the air surface of nano-biocomposite films can be comparable with values observed for corn starch films with the presence of oleic acid [27], however, such values are higher than those observed for alginate, alginate/chitosan and chitosan films [21], as well as, gelatin films containing Lap [17] and Mt [20]. Fig. 2. Image analysis was effective in quantifying the microstructure of nano-biocomposite films using SEM and AFM micrographs (Table 2). Different values were recorded for energy, entropy and 𝐹𝐷 using SEM and AFM micrographs. Such behavior was due to the difference in the magnification value used in each microscopy technique [21]. However, independent from the microscopy technique, texture parameters had the same tendency of surfaces and Lap concentration. Energy and entropy values on the support surface of nano-biocomposite films were not changed by the addition of Lap (p > 0.05). However, on the air surfaces, the
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energy values decreased and the entropy values increased, with Lap (p < 0.05) (Tables 2). The decreasing in energy values could be associated with an increase in the local heterogeneity of the pixels of the scanned image [17, 21]. This behavior was due probably to the distribution of Lap within amylose and amylopectin chains that lead to a decrease in surface uniformity [21]. Such increase in entropy by adding Lap could be correlated with the distribution of Lap platelets within amylose and amylopectin chains, increasing the randomness of images and the degree of local pixel variations of the image [21]. The large heterogeneity on the air surface of nanobiocomposite films was associated with a low energy and a high entropy value when compared with the support surface of each sample. Valencia et al. [17] and ArzateVázquez et al. [21] determined similar entropy values for nanocomposite films using gelatin-Lap, alginate and alginate/chitosan plasticized with glycerol and analyzed by AFM. For all nano-biocomposite films, 𝐹𝐷 values for the support surface decreased and for the air surface increased with Lap concentration (Table 2). According to ArzateVázquez et al. [21], 𝐹𝐷 is a measurement directly related to the degree of surface roughness. In this study, there was a relation between 𝐹𝐷 and roughness (𝑅𝑎 and 𝑅𝑞 ). Hence, the surfaces of nano-biocomposite films that showed the highest values for roughness had the highest 𝐹𝐷 values. 𝐹𝐷 values were in agreement with the values previously observed by Valencia et al. [17], Arzate-Vázquez et al. [21] and Ortiz-Zarama et al. [28] for 2-D gray-scale images of films based on gelatin-Lap, gelatin-carbon nanotubes, alginate and alginate/chitosan. 3.2.
Gloss and Color
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The gloss values of nano-biocomposite films depended upon the surfaces and the Lap concentration (Table 3). The support surface had high gloss values due to a low roughness (Table 1). For control films (0 g of Lap/100 g of CS), the gloss value for the support surface was approximately 73% higher when compared with the air surface (Table 3). These results are linked to the results of surfaces micrographs (Figs. 1 and 2). The increase in Lap concentration decreased the gloss values on the air surface of all nano-biocomposite films (Table 3). In contrast, no statistical differences were observed for gloss values on the support surface of nano-biocomposite films, varying Lap concentration (p > 0.05) (Table 3). This result is in accordance with a slight roughness change on the support surface with the addition of Lap, as observed previously during the AFM analyzes (Fig. 2 and Table 1). According to Trezza & Krochta [29], considering an angle of 60°, the materials that showed values higher than 70 units can be considered as having a high gloss, such as the films produced in this study (Table 3). The gloss of the films could be comparable to values observed for cassava starch films [30], nano-biocomposite films based on gelatin and Mt [20], and hydroxypropyl methylcellulose films [31]. However, they were higher than the values observed for pea starch and corn starch films [26, 32]. On the other hand, the color of nano-biocomposite films (∆𝐸 ∗ ) and the opacity were not affected by the Lap concentration, remaining below 3 and 1%, respectively, typical for films based on starches [26, 33]. Color and opacity results suggest that the nano-biocomposite films were colorless and completely transparent, due to exfoliation and/or intercalation of Lap platelets. 11
3.3.
Water contact angle
The values for water contact angle were changed according to surface and Lap concentration. No drastic modifications in water contact angles were recorded during the first 20 seconds of measurement for each nano-biocomposite film (Fig. 3). Therefore, we chose values at 15 seconds, after the drop touched the surface of the films, to compare the water contact angle of nano-biocomposite films (Table 3). In general, the support surface was more hydrophobic than the air surface of nanobiocomposite films (Table 3) probably due to changes in the surface roughness. Hence, by increasing the surface roughness, water contact angle decreased. The water contact angle of control films was similar to the values observed for cassava starch films plasticized with glycerol [30]. The incorporation of Lap nanoparticles into the starch matrix lead to a decrease in water contact angle (approximately 55%) on both surfaces (Table 3). According to Flaker et al. [20], the increase in the hydrophilicity of films based on biopolymers with nanoclays (Mt) can be due to an increase in the surface roughness of the samples. In this study, Lap increased and then decreased the surface roughness on the air and the support surfaces of nano-biocomposite films, respectively. However, both surfaces recorded a similar decrease in water contact angle (Table 3), suggesting that the surface roughness could be explained by hydrophilicity changes in nano-biocomposite films. These results suggested that the chemical structure of Lap, especially Na + ions, could increase the hydrophilicity of nano-biocomposite films [15]. Fig. 3.
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According to Vogler [34], values for water contact angle above 65° are typical of a hydrophobic surface; below 65°, they are typical of a hydrophilic surface. Thus, all nano-biocomposite films produced in this study can be classified as having hydrophilic surfaces (except the support surface of control films). 3.4.
X-ray diffraction spectra
X-ray diffraction (XRD) spectra of cassava starch (CS) and laponite (Lap) raw powders (Fig. 4a) were determined in order to compare those films. CS spectra had a diffraction pattern typical of an A-type crystal [35]. No diffraction peaks at 2θ ≤ 6.5° were observed for the CS powder. On the other hand, for the Lap powder, one distinct visible reflection appeared (2θ = 6.5°, d(001) = 14 Å) (Fig. 4a). This was related to the interlayer space of Lap platelets [36]. Fig. 4. All nano-biocomposite films had a XRD spectra typical of amorphous materials (Fig. 4b) [11]. The presence of Lap in the films influenced the XRD spectra at 2θ ≤ 6.5° (insert in Fig. 4b). For films containing 1.5 and 3 g of Lap/100 g of CS, the XRD spectrum revealed a complete exfoliation of Lap platelets. No diffraction peaks were observed at 2θ ≤ 6.5°, suggesting that Lap platelets are at least 44 Å apart (2θ = 2°). Rao [37] observed that at an exfoliated state, the reflection of the interlayer space detected at 2θ = 6.5° in the Lap raw powder disappears as a consequence of that interlayer space being open. When the Lap concentration was higher than 3 g of Lap/100 g of CS, a diffraction peak at 2θ = 5.2° (d(001)= 17 Å) was observed, suggesting the presence of an intercalated Lap between starch chains. X-ray results showed that the exfoliation and/or intercalation of Lap was achieved by nano-biocomposite films. 13
4. Conclusions The laponite concentration (≤ 6 g of Lap/100 g of CS) was associated with drastic morphological modifications in nano-biocomposite films. Nano-biocomposite films produced by casting presented differences in surface roughness on the support and the air surfaces. Texture image analysis using scanning electron microscopy and atomic force microscopy were successful to describe quantitatively the microsructure of nanobiocomposite films. Changes in energy and entropy values were associated with the surface heterogeneity. The fractal dimension was associated with changes in roughness and optical propeties (gloss) on the surfaces of nano-biocomposite films. Nano-biocomposite films added with laponite were colorless and completely transparent because of exfoliation and/or intercalation of laponite platelets, similar to control films (without laponite). Surface roughness may change the properties of hidrophobic surfaces of nanobiocomposite films. Hence, the support surface was more hydrophobic than the air surface. Laponite increased hydrophicity in both surfaces probalby due to Na+ ions inside the laponite structure. Acknowledgments The authors gratefully acknowledge to São Paulo Research Foundation (FAPESP), for the PhD fellowship of the first author (2012/24047-3), and for the grant (2013/07914-8). References [1] A.S. Abre, M. Oliveira, A. Sá, R.M. Rodrigues, M.A. Cerqueira, A. Vicente, A.V. Machado. Antimicrobial nanostructured starch based films for packaging. 14
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[23] P. Bergo, P.J.A. Sobral, V.C. Guevara, A.C. Vadala. Semi crystalline behaviour of cassava starch films plasticized with glycerol. Mater Sci Forum, 636–637 (2010), pp. 745–752. [24] L. Tang, S. Chen, W. Su, W. Weng, K. Osako, M. Tanaka. Physicochemical properties and film-forming ability of fish skin collagen extracted from different freshwater species. Process Biochem., 50 (1) (2015), pp. 148–155. [25] K. Okeyoshi, M.K. Okajima, T. Kaneko. Milliscale Self-Integration of Megamolecule Biopolymers on a Drying Gas–Aqueous Liquid Crystalline Interface. Biomacromolecules, 17 (6) (2016), pp. 2096–2103. [26] A. Cano, E. Fortunati, M. Cháfer, C. González-Martínez, A. Chiralt, J.M. Kenny. Effect of cellulose nanocrystals on the properties of pea starch–poly (vinyl alcohol) blend films. J Mater Sci., 50 (21) (2015), pp. 6979–6992. [27] A. Jiménez, M.J. Fabra, P. Talens, A. Chiralt. Effect of re-crystallization on tensile, optical and water vapour barrier properties of corn starch films containing fatty acids. Food Hydrocoll., 26 (2012), pp. 302–310. [28] M.A. Ortiz-Zarama, A. Jiménez-Aparicio, M.J. Perea-Flores, J. Solorza-Feria. Barrier, mechanical and morpho-structural properties of gelatin films with carbon nanotubes addition. J Food Eng., 120 (2014), pp. 223–232. [29] T.A. Trezza, J.M. Krochta. The gloss of edible coatings as affected by surfactants, lipids, relative humidity, and time. J Food Sci., 65 (4) (2000), pp. 658–662. [30] C. Bangyekan, D. Aht-Ong, K. Srikulkit. Preparation and properties evaluation of chitosan-coated cassava starch films. Carbohyd Polym., 63 (1) (2006), pp. 61–71. 18
[31] R. Villalobos, J. Chanona, P. Hernández, G. Gutiérrez, A. Chiralt. Gloss and transparency of hydroxypropyl methylcellulose films containing surfactants as affected by their microstructure. Food Hydrocoll., 19 (1) (2005), pp. 53–61. [32] R. Ortega-Toro, J. Contreras, P. Talens, A. Chiralt. Physical and structural properties and thermal behaviour of starch-poly (e-caprolactone) blend films for food packaging. Food Packaging and Shelf Life, 5 (2015), pp. 10–20. [33] P. Veiga-Santos, C.K. Suzuki, M.P. Cereda, A.R.P. Scamparini. Microstructure and color of starch-gum films: Effect of gum deacetylation and additives. Part 2. Food Hydrocoll., 19 (6) (2005), pp. 1064–1073. [34] E.A. Vogler. Structure and reactivity of water at biomaterial surfaces. Adv Colloid Interface Sci., 74 (1–3) (1998), pp. 69–117. [35] G.A. Valencia, M. Djabourov, P.J.A. Sobral. Water desorption of cassava starch granules: A study based on thermogravimetric analysis of aqueous suspensions and humid powders. Carbohyd Polym., 147 (2016), pp. 533–541. [36] L. Bippus, M. Jaber, B. Lebeau. Laponite and hybrid surfactant/laponite particles processed as spheres by spray-drying. New J Chem, 33 (5) (2009), pp. 1116–1126. [37] Y. Rao. Gelatin-clay nanocomposites of improved properties, Polymers, 48 (18) (2007), pp. 5369–5375.
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Figures Captions Fig. 1. Examples of scanning electron micrographs of nano-biocomposite films with laponite concentration of 0 (a,b), 3 (c,d), and 6 g of Lap/100 g of CS, (e,f). Support surface (left) and air surface (right). Fig. 2. Examples of atomic force micrographs of nano-biocomposite films with laponite concentration of 0 (a,b), 3 (c,d), and 6 g of Lap/100 g of CS (e,f). Support surface (left) and air surface (right). Fig. 3. Examples of water contact angle of nano-biocomposite films: air (a) and support (b) surfaces. Concentrations of laponite are presented inside figures as g of Lap/100 g of CS. Fig. 4. X-ray diffraction spectra of laponite and cassava starch powders (a) and nano-biocomposite films (b). Concentrations of laponite are presented inside figures as g of Lap/100 g of CS.
20
Fig. 1.
21
Fig. 2.
22
Fig. 3.
Fig. 4.
23
Table 1. Average roughness (𝑅𝑎 ) and root mean square roughness (𝑅𝑞 ) of nanobiocomposite films with different laponite concentrations and for the two surfaces (support and air). Lap concentration 𝑹𝒂 (nm) (g/100 g of CS) Support 0 12 ± 2A,b 1.5 5 ± 1B,b 3 5 ± 0B,b 4.5 5 ± 0B,b 6 6 ± 1B,b Lap = laponite. CS = cassava starch.
𝑹𝒒 (nm) Air 111 ± 6B,a 119 ± 5B,a 138 ± 10AB,a 140 ± 19AB,a 161 ± 26A,a
Support 15 ± 3A,b 7 ± 1B,b 7 ± 0B,b 7 ± 1B,b 8 ± 1B,b
Air 133 ± 18B,a 151 ± 7B,a 170 ± 14AB,a 174 ± 25AB,a 200 ± 32A,a
All values were expressed as mean ± standard error (n = 5). Means in the same column followed by the same capital letter are not significantly different (p < 0.05). Means in the same line followed by the same lowercase are not significantly different (p < 0.05).
24
Table 2. Textural parameters (energy, entropy and fractal dimension, 𝐹𝐷) calculated from scanning electron microscopy and atomic force microscopy micrographs of nano-biocomposite films with different laponite concentrations and for the two surfaces (support and air). SEM micrographs Lap Energy (x104) concentration (g/100 g of CS) Support Air 30.0 ± 22.0 0 0.1a 0.4A,b 30.0 ± 21.0 1.5 0.1a 0.1B,b 30.0 ± 21.0 3 0.1a 0.1B,b 30.0 ± 20.0 4.5 0.1a 0.1C,b 30.0 ± 20.0 a 6 0.1 0.1C,b
Entropy
FD*
Support
Air
Support
Air
6.0 ± 0.0b
6.3 ± 0.1B,a
6.0 ± 0.0b
6. 5 ± 0.1B,a
2.3 ± 0.0a
2.3 ± 0.0B,a
6.1 ± 0.1b
6.6 ± 0.0A,a
2.3 ± 0.0b
2.4 ± 0.0A,a
6.1 ± 0.1b
6.6 ± 0.0A,a
2.2 ± 0.1b
2.4 ± 0.0A,a
6.6 ± 0.0A,a 2.2 ± 0.1b AFM micrographs
2.4 ± 0.0A,a
± 2.3 ± 0.0a 2.3 ± 0.0B,a
± ± ± ±
6.2 ± a 0 0.1 4.3 ± 0.1A,b 6.2 ± 4.1 ± a AB,b 1.5 0.1 0.1 6.1 ± 3.9 ± a AB,b 3 0.1 0.1 6.1 ± 4.5 0.1a 3.5 ± 0.1C,b 6.1 ± 6 0.1a 3.4 ± 0.2C,b Lap = laponite. CS = cassava starch.
6.1 ± 0.1b
7.7 ± 0.2b
8.4 ± 0.0C,a
2.6 ± 0.0A,a
2.6 ± 0.0B,a
7.7 ± 0.1b
8. 6 ± 0.1B,a
2.6 ± 0.0A,a
2.6 ± 0.0B,a
7.7 ± 0.1b
8.7 ± 0.1B,a
2.6 ± 0.0A,a
2.6 ± 0.0B,a
7.9 ± 0.2b
8.8 ± 0.1AB,a
2.5 ± 0.0B,b
2.7 ± 0.0A,a
8.0 ± 0.2b
8.9 ± 0.0A,a
2.5 ± 0.0B,b
2.7 ± 0.0A,a
All values were expressed as mean ± standard error (n = 5). *Regression coefficients of the straight lines obtained from the log(box count) vs. log(box size) plot were higher than 0.991, in scanning electron microscopy micrographs, and higher than 0.992, in atomic force microscopy micrographs.
25
Means in the same column followed by the same capital letter are not significantly different (p < 0.05). Means in the same line followed by the same lowercase are not significantly different (p < 0.05).
26
Table 3. Gloss and water contact angle of nano-biocomposite films with different laponite concentrations and for the two surfaces (support and air). Lap Gloss (U.B.) concentration (g/100 g of CS) Support 0 93.8 ± 4.5ª 1.5 87.1 ± 4.1ª 3 87.1 ± 4.1ª 4.5 87.4 ± 5.4ª 6 89.3 ± 5.5a Lap = laponite. CS = cassava starch.
Water contact angle (°) Air 54.1 ± 3.9A,b 45.7 ± 4.6B,b 44.6 ± 4.4B,b 38.5 ± 5.1BC,b 33.5 ± 3.8C,b
Support 75.4 ± 4.6A,a 56.3 ± 7.1B,a 50.1 ± 1.7B,a 46.8 ± 5.7BC,a 42.1 ± 0.2C,a
Air 55.3 ± 0.1A,b 42.7 ± 5.1B,b 29.8 ± 0.7C,b 32.4 ± 2.1C,b 29.9 ± 3.9C,b
All values were expressed as mean ± standard error (n = 3). Means in the same column followed by the same capital letter are not significantly different (p < 0.05). Means in the same line followed by the same lowercase are not significantly different (p < 0.05).
27
S0141-8130(17)33048-9 https://doi.org/10.1016/j.ijbiomac.2017.10.031 BIOMAC 8326
To appear in:
International Journal of Biological Macromolecules
Received date: Accepted date:
15-8-2017 5-10-2017
Please cite this article as: Germ´an Ayala Valencia, Carla Giovana Luciano, Rodrigo Vinicius Lourenc¸o, Paulo Jos´e do Amaral Sobral, Microstructure and physical properties of nano-biocomposite films based on cassava starch and laponite, International Journal of Biological Macromolecules https://doi.org/10.1016/j.ijbiomac.2017.10.031 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.
Microstructure and physical properties of nano-biocomposite films based on cassava starch and laponite Germán Ayala Valencia*, Carla Giovana Luciano, Rodrigo Vinicius Lourenço, Paulo José do Amaral Sobral**. Department of Food Engineering, Faculty of Animal Science and Food Engineering, University of São Paulo, Pirassununga, SP, Brazil. *Corresponding *[email protected]; **[email protected] Abstract The aim of this research was to study the effects of laponite concentrations on some properties of nano-biocomposite films based on cassava starch, focusing mainly the relation between the properties of the surface microstructure and roughness, water contact angle and gloss. Nano-biocomposite films were produced by casting. We analyzed gloss, color, opacity, water contact angle, crystallinity by X-ray diffraction, and microstructure by scanning electron microscopy and atomic force microscopy. Texture parameters (energy, entropy and fractal dimension) were extracted from micrographs. We observed a great impact of laponite in the morphology of nanobiocomposite films. Texture parameters correlated with surface heterogeneity and roughness. Finally, surface roughness affected the surface hydrophilicity of nanobiocomposite films. Laponite platelets were exfoliated and/or intercalated with amylose and amylopectin chains. This research reports new information on the effects of laponite concentrations on the morphological, optical and wetting properties of nano-biocomposite films aiming future industrial applications.
1
Keywords: gloss; image analysis; nano-biocomposite films; microstructure; water contact angle. 1. Introduction The development of nanostructured materials has a promising advance in several areas such as food packaging. Normally, such nanostructured materials are composed by a natural polymer matrix, called biopolymer, and an organic/inorganic particle loading, which are characterized by having at least one dimension between 1 and 100 nm [1, 2]. Starch is a good candidate for the biopolymer matrix of nanostructured materials because it is cheap, abundant, renewable, biocompatible and biodegradable [1], however, films based on starch present poor mechanical properties and a high water sensibility [3, 4]. Several methods have been studied to avoid or reduce this behavior such as the use of different plasticizers [4, 5], blending starch with a biodegradable synthetic polymer such as poly(vinyl alcohol) [6], or even the use of flours [7], which are natural blends of starch with proteins [8]. All cases resulted in a relative success regarding the improvement of the overall properties of films. A recent alternative to improve the physical properties of starch films is a reinforcement with nanoclays [1, 9-12], especially montmorillonite (Mt), which has been the nanoclay most studied for starch films [1, 9-11, 13]. Another nanoclay not commonly studied for starch-based films is laponite (Lap) [2, 9, 11]. Lap (hydrous sodium lithium magnesium silicate) is a crystalline layer silicate with a structure and a composition that closely resembles smectite (Sm) [14]. Its chemical formula is Si 8[Mg5.5Li0.4H4.0O24.0]0.7-[Na0.7]0.7+ [15]. The Lap nanoparticle has
2
the shape of a disk with a thickness of approximately 1 nm and a diameter of approximately 25 nm, being smaller than raw clays such as Mt [14]. Lap has been successfully dispersed into cellulose and gelatin matrixes, improving the mechanical and water vapor permeability properties without changing the optical properties of nano-biocomposite films [16-18]. Moreover, some starch nanobiocomposite films have been developed using the casting technique [2, 9, 11]. However, no study explored the relation between the surface microstructure and the surface properties of films affected by different Lap concentrations. Thus, the objective of this research was to study the effects of laponite concentrations on physical properties of nano-biocomposite films based on cassava starch, specially the relation between the surface microstructure and other surfaces (roughness, water contact angle and gloss) properties. 2. Materials and methods 2.1.
Materials
Cassava starch was purchased from Yoki Food Industry (São Paulo, Brazil) and was used as the biopolymer. Glycerol (Synth, Brazil) was used as a plasticizer, and laponite RD (Southerm Clay Products Inc. Reference number 23224) was used as the nanoparticle. The initial water content in cassava starch, glycerol and Lap was 12.4 ± 0.1, 14.1 ± 0.1 and 7.5 ± 0.1% (wet basis), respectively. Distilled water was used as the solvent. 2.2.
Preparation and characterization of nano-biocomposite films
Nano-biocomposite films were produced using a mixture of cassava starch (CS) (solution A) and laponite (Lap) dispersed into water containing glycerol (solution B). Solution A was prepared using 4 g of CS/100 g of solution, as follows: cassava starch 3
was heated at 90 ± 1°C for 30 min to gelatinize starch [19]. At the same time, solution B was prepared at room temperature using 1 g of Lap/100 g solution in distilled water and a high-speed homogenizer (ultraturrax, Ika, model T25) at 20,000 rpm for 30 min [19]. Then, glycerol (25 g of glycerol/100 g of CS) was added to the Lap dispersion and mixed for 15 min using a magnetic stirrer (Tecnal TE 0852, Brazil). Then, the solutions A and B were mixed conveniently to produce solutions with 0, 1.5, 3, 4.5 and 6 g of Lap/100 g of CS. After, the solutions were homogenized for 15 min at 90 ± 1°C using the same thermostatic water-bath. Nano-biocomposite films were produced by casting. The amount of solution poured into Petri dishes (diameter: 14 cm) was calculated to obtain a constant weight of dry matter of approximately 8 mg/cm2. All solutions were dried in a forced-air circulation oven (Marconi, MA037, Brazil) at 30°C and controlled relative humidity (RH = 60 ± 5%) for 20 hours. Nano-biocomposite films have a thickness of 80 ± 10 µm. For the SEM (scanning electron microscopy), AFM (atomic force microscopic) and X-ray diffraction analyses, samples were previously conditioned over silica gel, into desiccators for at least 7 days. For the characterization of gloss, color and contact angle, samples were conditioned into desiccators containing saturated solutions of Mg(NO3)2 (RH = 50.3%) at 25°C for at least 7 days. 2.2.1. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) The air and support surfaces of the nano-biocomposite films were fixed to aluminum stubs using a double-sided tape. The micrographs were taken at sample random positions with a 1000x magnification, using a scanning electron microscopy (SEM model TM-3000, Hitachi), at 5 kV [20]. The resolution of the micrographs was 0.13 4
µm/px and the field of view was 0.021 mm2. Five micrographs were made for each surface (air and support surfaces) for every nano-biocomposite film. The nano-biocomposite films were analyzed using an atomic force microscope (model NT-MDT Solver Next Brand, Russia), equipped with a software for image analysis. The nano-biocomposite films were analyzed using the semi-contact mode with a resonance frequency of 150 kHz, a contact force of 5 N/m and a scan speed 0.3 Hz [17]. Analyses were performed in 50 µm x 50 µm areas (field of view: 0.0025 mm2) using five different samples. The treatment of the images was performed using second-order subtraction and a software for images analysis (New Model 3.1.0 program PX). The average roughness (𝑅𝑎 ) and the root mean square roughness (𝑅𝑞 ) were calculated using the AFM software. All micrographs were made using RGB colors with a resolution of 0.2 µm/px. Texture image analysis was performed to quantify the surface microstructure captured by SEM and AFM micrographs. Three texture parameters (energy, entropy and fractal dimension) were analyzed using the software ImageJ v 1.39 (National Institute Health, Bethesda, MD, USA). Energy and entropy were determined in grayscale using the Gray Level Co-Ocurrence Matrix algorithm (GLCM) by fixing the distance of the pixels (d) to 1 and the angle of displacement (θ) to 0° [17, 21]. The fractal dimension (𝐹𝐷) was calculated based on gray-scale images using power-law scaling and Standard Box Count method [17, 21]. 𝐹𝐷 values were calculated taking into account the slope in the log(box count) vs. log(box size) plot, according to Arzate-Vázquez et al. [21]. 2.2.2. Gloss
5
The gloss of nano-biocomposite films was determined using a gloss meter (Rhodopoint NGL 20/60), at angle of 60° in 10 points of each sample according to the ASTM D2457 standard [20]. Gloss measurements were performed on the air and the support surfaces of the nano-biocomposite films. 2.2.3. Films color and Opacity The color of nano-biocomposite films was determined using a Miniscan XE colorimeter (HunterLab), in the reflectance mode adopting the CIELab scale, an illuminant/angle D65/10° and an opening of 30 mm [17]. Samples were placed on the surface of the standard white plate and the parameters 𝐿∗ (lightness index), 𝑎∗ (color tonalities from red to green) and 𝑏∗ (color tonalities from yellow to blue) were determined. The total color difference (∆𝐸 ∗ ) was then calculated as: ∆𝐸 ∗ = √(∆𝐿∗ )2 + (∆𝑎∗ )2 + (∆𝑏∗ )2
(1)
∗ ∗ where∆𝐿∗ = 𝐿∗𝑠𝑎𝑚𝑝𝑙𝑒 - 𝐿∗𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 (92.58 ± 0.92); ∆𝑎∗ = 𝑎𝑠𝑎𝑚𝑝𝑙𝑒 - 𝑎𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 (-0.88 ± 0.06); ∗ ∗ ∆𝑏∗ = 𝑏𝑠𝑎𝑚𝑝𝑙𝑒 - 𝑏𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 (1.26 ± 0.02).
Opacity was measured using the same equipment of the color measurement, according to the Hunterlab method, in reflectance mode. Opacity (𝑌) values were 𝑌
calculated according the 𝑌 = 𝑌 𝑏 relation between the opacity of nano-biocomposite 𝑊
films superimposed over the black standard (𝑌𝑏 ) and the same sample superimposed over the white standard (𝑌𝑊 ). 2.2.4. Water contact angle The water contact angle of nano-biocomposite films was measured according to the ASTM D7334 standard and using an optical tensiometer (Attension Theta lite, KSV Instrument) [22]. The nano-biocomposite films were attached to the equipment and 6
one drop of distilled water of approximate 10 µL was dropped over the sample using an automatic precision syringe. The preparation was immediately photographed. The angle formed between the sample surface and the tangent of the drop was calculated using the software Attension Theta Lite. The measurements of water contact angle were made by analyzing the shape of one sessile drop after it had been placed over the samples for 30 seconds. The measurements of water contact angle were made on the air and the support surfaces of nano-biocomposite films. 2.2.5. X-ray diffraction X-ray diffractograms (XRD) of nano-biocomposite films were obtained using an Xray diffractometer (AXS Analytical X-ray Systems Siemens D 5005, Germany), operating at 40 kV and 40 mA (Cu Kα 1, λ = 1.54056 Å radiation). Samples (3 cm x 3 cm) were placed inside an aluminum frame. The spectra were recorded at 25°C between the angles 2θ = 2° and 50° at a rate of 2°/min [23]. Samples of Lap and cassava starch powders were also analyzed under the same conditions. The reflection peak, which is typical of Lap layers, was determined as 𝑑(001) applying the Bragg law (Equation 2) to the Lap spectrum and some films spectra whenever visible. 𝜆 = 2𝑑sinθ
(2)
where 𝜆 is the wavelength of Cu Kα radiation; 𝑑 is the spacing between Lap layers, and θ is the reflection angle. 2.3.
Statistical analyses
7
Analysis of variance (ANOVA) and a Tukey test with multiple comparisons were performed with a significance level of 5% using the ‘‘Statistical Analysis Systems’’ software (version 9.2, SAS). 3. Results and discussions 3.1.
Films morphological properties
The support surfaces were smoother than the air surfaces of nano-biocomposite films (Fig. 1). Similar results were observed by Tang et al. [24] and Okeyoshi et al. [25], who studied films based on collagen and xanthan gum, respectively. The Lap concentration increased the air surface roughness of nano-biocomposite films. Big structures were observed in films containing 6 g of Lap/100 g of CS (see red arrows in Fig. 1f) probably due to the biopolymer aggregation. According to Cano et al. [26], nanoparticles could induce a phase separation among biopolymers of nanostructured films, promoting aggregations.
Fig. 1. AFM micrographs of films confirmed that the support surface roughness was lower than in the air surface (Fig. 2), as previously evidenced by the SEM micrographs (Fig. 1). Roughness values (𝑅𝑎 and 𝑅𝑞 ) confirmed the observations using SEM and AFM micrographs (Table 1): for control films (0 g of Lap/100 g of CS), 𝑅𝑎 and 𝑅𝑞 values in air surfaces were almost nine times higher than the 𝑅𝑎 and 𝑅𝑞 values recorded on the support surface. For control films, roughness values on the air surface were similar to those observed by Jiménez et al. [27], who studied corn starch films plasticized with glycerol.
8
The roughness on the support surface decreased upon adding Lap from 12 to 6 nm (𝑅𝑎 ) and from 15 to 8 nm (𝑅𝑞 ), when the Lap concentration increased from 0 to 6 g of Lap/100 g of CS. Smoother surfaces added with Lap could be a consequence of biopolymer orientation on the support surface during the drying process. An opposite behavior was observed for the roughness on the air surface: 𝑅𝑎 increased from 112 to 161 nm and 𝑅𝑞 increased from 133 to 200 nm, when the Lap concentration increased from 0 to 6 g of Lap/100 g of CS. That is, the presence of Lap increased the roughness on the air surface of nano-biocomposite films probably due to the formation of agglomerates because of the interaction biopolymer chains–Lap platelets during the drying process of filmogenic solutions [20]. 𝑅𝑎 and 𝑅𝑞 values on the air surface of nano-biocomposite films can be comparable with values observed for corn starch films with the presence of oleic acid [27], however, such values are higher than those observed for alginate, alginate/chitosan and chitosan films [21], as well as, gelatin films containing Lap [17] and Mt [20]. Fig. 2. Image analysis was effective in quantifying the microstructure of nano-biocomposite films using SEM and AFM micrographs (Table 2). Different values were recorded for energy, entropy and 𝐹𝐷 using SEM and AFM micrographs. Such behavior was due to the difference in the magnification value used in each microscopy technique [21]. However, independent from the microscopy technique, texture parameters had the same tendency of surfaces and Lap concentration. Energy and entropy values on the support surface of nano-biocomposite films were not changed by the addition of Lap (p > 0.05). However, on the air surfaces, the
9
energy values decreased and the entropy values increased, with Lap (p < 0.05) (Tables 2). The decreasing in energy values could be associated with an increase in the local heterogeneity of the pixels of the scanned image [17, 21]. This behavior was due probably to the distribution of Lap within amylose and amylopectin chains that lead to a decrease in surface uniformity [21]. Such increase in entropy by adding Lap could be correlated with the distribution of Lap platelets within amylose and amylopectin chains, increasing the randomness of images and the degree of local pixel variations of the image [21]. The large heterogeneity on the air surface of nanobiocomposite films was associated with a low energy and a high entropy value when compared with the support surface of each sample. Valencia et al. [17] and ArzateVázquez et al. [21] determined similar entropy values for nanocomposite films using gelatin-Lap, alginate and alginate/chitosan plasticized with glycerol and analyzed by AFM. For all nano-biocomposite films, 𝐹𝐷 values for the support surface decreased and for the air surface increased with Lap concentration (Table 2). According to ArzateVázquez et al. [21], 𝐹𝐷 is a measurement directly related to the degree of surface roughness. In this study, there was a relation between 𝐹𝐷 and roughness (𝑅𝑎 and 𝑅𝑞 ). Hence, the surfaces of nano-biocomposite films that showed the highest values for roughness had the highest 𝐹𝐷 values. 𝐹𝐷 values were in agreement with the values previously observed by Valencia et al. [17], Arzate-Vázquez et al. [21] and Ortiz-Zarama et al. [28] for 2-D gray-scale images of films based on gelatin-Lap, gelatin-carbon nanotubes, alginate and alginate/chitosan. 3.2.
Gloss and Color
10
The gloss values of nano-biocomposite films depended upon the surfaces and the Lap concentration (Table 3). The support surface had high gloss values due to a low roughness (Table 1). For control films (0 g of Lap/100 g of CS), the gloss value for the support surface was approximately 73% higher when compared with the air surface (Table 3). These results are linked to the results of surfaces micrographs (Figs. 1 and 2). The increase in Lap concentration decreased the gloss values on the air surface of all nano-biocomposite films (Table 3). In contrast, no statistical differences were observed for gloss values on the support surface of nano-biocomposite films, varying Lap concentration (p > 0.05) (Table 3). This result is in accordance with a slight roughness change on the support surface with the addition of Lap, as observed previously during the AFM analyzes (Fig. 2 and Table 1). According to Trezza & Krochta [29], considering an angle of 60°, the materials that showed values higher than 70 units can be considered as having a high gloss, such as the films produced in this study (Table 3). The gloss of the films could be comparable to values observed for cassava starch films [30], nano-biocomposite films based on gelatin and Mt [20], and hydroxypropyl methylcellulose films [31]. However, they were higher than the values observed for pea starch and corn starch films [26, 32]. On the other hand, the color of nano-biocomposite films (∆𝐸 ∗ ) and the opacity were not affected by the Lap concentration, remaining below 3 and 1%, respectively, typical for films based on starches [26, 33]. Color and opacity results suggest that the nano-biocomposite films were colorless and completely transparent, due to exfoliation and/or intercalation of Lap platelets. 11
3.3.
Water contact angle
The values for water contact angle were changed according to surface and Lap concentration. No drastic modifications in water contact angles were recorded during the first 20 seconds of measurement for each nano-biocomposite film (Fig. 3). Therefore, we chose values at 15 seconds, after the drop touched the surface of the films, to compare the water contact angle of nano-biocomposite films (Table 3). In general, the support surface was more hydrophobic than the air surface of nanobiocomposite films (Table 3) probably due to changes in the surface roughness. Hence, by increasing the surface roughness, water contact angle decreased. The water contact angle of control films was similar to the values observed for cassava starch films plasticized with glycerol [30]. The incorporation of Lap nanoparticles into the starch matrix lead to a decrease in water contact angle (approximately 55%) on both surfaces (Table 3). According to Flaker et al. [20], the increase in the hydrophilicity of films based on biopolymers with nanoclays (Mt) can be due to an increase in the surface roughness of the samples. In this study, Lap increased and then decreased the surface roughness on the air and the support surfaces of nano-biocomposite films, respectively. However, both surfaces recorded a similar decrease in water contact angle (Table 3), suggesting that the surface roughness could be explained by hydrophilicity changes in nano-biocomposite films. These results suggested that the chemical structure of Lap, especially Na + ions, could increase the hydrophilicity of nano-biocomposite films [15]. Fig. 3.
12
According to Vogler [34], values for water contact angle above 65° are typical of a hydrophobic surface; below 65°, they are typical of a hydrophilic surface. Thus, all nano-biocomposite films produced in this study can be classified as having hydrophilic surfaces (except the support surface of control films). 3.4.
X-ray diffraction spectra
X-ray diffraction (XRD) spectra of cassava starch (CS) and laponite (Lap) raw powders (Fig. 4a) were determined in order to compare those films. CS spectra had a diffraction pattern typical of an A-type crystal [35]. No diffraction peaks at 2θ ≤ 6.5° were observed for the CS powder. On the other hand, for the Lap powder, one distinct visible reflection appeared (2θ = 6.5°, d(001) = 14 Å) (Fig. 4a). This was related to the interlayer space of Lap platelets [36]. Fig. 4. All nano-biocomposite films had a XRD spectra typical of amorphous materials (Fig. 4b) [11]. The presence of Lap in the films influenced the XRD spectra at 2θ ≤ 6.5° (insert in Fig. 4b). For films containing 1.5 and 3 g of Lap/100 g of CS, the XRD spectrum revealed a complete exfoliation of Lap platelets. No diffraction peaks were observed at 2θ ≤ 6.5°, suggesting that Lap platelets are at least 44 Å apart (2θ = 2°). Rao [37] observed that at an exfoliated state, the reflection of the interlayer space detected at 2θ = 6.5° in the Lap raw powder disappears as a consequence of that interlayer space being open. When the Lap concentration was higher than 3 g of Lap/100 g of CS, a diffraction peak at 2θ = 5.2° (d(001)= 17 Å) was observed, suggesting the presence of an intercalated Lap between starch chains. X-ray results showed that the exfoliation and/or intercalation of Lap was achieved by nano-biocomposite films. 13
4. Conclusions The laponite concentration (≤ 6 g of Lap/100 g of CS) was associated with drastic morphological modifications in nano-biocomposite films. Nano-biocomposite films produced by casting presented differences in surface roughness on the support and the air surfaces. Texture image analysis using scanning electron microscopy and atomic force microscopy were successful to describe quantitatively the microsructure of nanobiocomposite films. Changes in energy and entropy values were associated with the surface heterogeneity. The fractal dimension was associated with changes in roughness and optical propeties (gloss) on the surfaces of nano-biocomposite films. Nano-biocomposite films added with laponite were colorless and completely transparent because of exfoliation and/or intercalation of laponite platelets, similar to control films (without laponite). Surface roughness may change the properties of hidrophobic surfaces of nanobiocomposite films. Hence, the support surface was more hydrophobic than the air surface. Laponite increased hydrophicity in both surfaces probalby due to Na+ ions inside the laponite structure. Acknowledgments The authors gratefully acknowledge to São Paulo Research Foundation (FAPESP), for the PhD fellowship of the first author (2012/24047-3), and for the grant (2013/07914-8). References [1] A.S. Abre, M. Oliveira, A. Sá, R.M. Rodrigues, M.A. Cerqueira, A. Vicente, A.V. Machado. Antimicrobial nanostructured starch based films for packaging. 14
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Figures Captions Fig. 1. Examples of scanning electron micrographs of nano-biocomposite films with laponite concentration of 0 (a,b), 3 (c,d), and 6 g of Lap/100 g of CS, (e,f). Support surface (left) and air surface (right). Fig. 2. Examples of atomic force micrographs of nano-biocomposite films with laponite concentration of 0 (a,b), 3 (c,d), and 6 g of Lap/100 g of CS (e,f). Support surface (left) and air surface (right). Fig. 3. Examples of water contact angle of nano-biocomposite films: air (a) and support (b) surfaces. Concentrations of laponite are presented inside figures as g of Lap/100 g of CS. Fig. 4. X-ray diffraction spectra of laponite and cassava starch powders (a) and nano-biocomposite films (b). Concentrations of laponite are presented inside figures as g of Lap/100 g of CS.
20
Fig. 1.
21
Fig. 2.
22
Fig. 3.
Fig. 4.
23
Table 1. Average roughness (𝑅𝑎 ) and root mean square roughness (𝑅𝑞 ) of nanobiocomposite films with different laponite concentrations and for the two surfaces (support and air). Lap concentration 𝑹𝒂 (nm) (g/100 g of CS) Support 0 12 ± 2A,b 1.5 5 ± 1B,b 3 5 ± 0B,b 4.5 5 ± 0B,b 6 6 ± 1B,b Lap = laponite. CS = cassava starch.
𝑹𝒒 (nm) Air 111 ± 6B,a 119 ± 5B,a 138 ± 10AB,a 140 ± 19AB,a 161 ± 26A,a
Support 15 ± 3A,b 7 ± 1B,b 7 ± 0B,b 7 ± 1B,b 8 ± 1B,b
Air 133 ± 18B,a 151 ± 7B,a 170 ± 14AB,a 174 ± 25AB,a 200 ± 32A,a
All values were expressed as mean ± standard error (n = 5). Means in the same column followed by the same capital letter are not significantly different (p < 0.05). Means in the same line followed by the same lowercase are not significantly different (p < 0.05).
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Table 2. Textural parameters (energy, entropy and fractal dimension, 𝐹𝐷) calculated from scanning electron microscopy and atomic force microscopy micrographs of nano-biocomposite films with different laponite concentrations and for the two surfaces (support and air). SEM micrographs Lap Energy (x104) concentration (g/100 g of CS) Support Air 30.0 ± 22.0 0 0.1a 0.4A,b 30.0 ± 21.0 1.5 0.1a 0.1B,b 30.0 ± 21.0 3 0.1a 0.1B,b 30.0 ± 20.0 4.5 0.1a 0.1C,b 30.0 ± 20.0 a 6 0.1 0.1C,b
Entropy
FD*
Support
Air
Support
Air
6.0 ± 0.0b
6.3 ± 0.1B,a
6.0 ± 0.0b
6. 5 ± 0.1B,a
2.3 ± 0.0a
2.3 ± 0.0B,a
6.1 ± 0.1b
6.6 ± 0.0A,a
2.3 ± 0.0b
2.4 ± 0.0A,a
6.1 ± 0.1b
6.6 ± 0.0A,a
2.2 ± 0.1b
2.4 ± 0.0A,a
6.6 ± 0.0A,a 2.2 ± 0.1b AFM micrographs
2.4 ± 0.0A,a
± 2.3 ± 0.0a 2.3 ± 0.0B,a
± ± ± ±
6.2 ± a 0 0.1 4.3 ± 0.1A,b 6.2 ± 4.1 ± a AB,b 1.5 0.1 0.1 6.1 ± 3.9 ± a AB,b 3 0.1 0.1 6.1 ± 4.5 0.1a 3.5 ± 0.1C,b 6.1 ± 6 0.1a 3.4 ± 0.2C,b Lap = laponite. CS = cassava starch.
6.1 ± 0.1b
7.7 ± 0.2b
8.4 ± 0.0C,a
2.6 ± 0.0A,a
2.6 ± 0.0B,a
7.7 ± 0.1b
8. 6 ± 0.1B,a
2.6 ± 0.0A,a
2.6 ± 0.0B,a
7.7 ± 0.1b
8.7 ± 0.1B,a
2.6 ± 0.0A,a
2.6 ± 0.0B,a
7.9 ± 0.2b
8.8 ± 0.1AB,a
2.5 ± 0.0B,b
2.7 ± 0.0A,a
8.0 ± 0.2b
8.9 ± 0.0A,a
2.5 ± 0.0B,b
2.7 ± 0.0A,a
All values were expressed as mean ± standard error (n = 5). *Regression coefficients of the straight lines obtained from the log(box count) vs. log(box size) plot were higher than 0.991, in scanning electron microscopy micrographs, and higher than 0.992, in atomic force microscopy micrographs.
25
Means in the same column followed by the same capital letter are not significantly different (p < 0.05). Means in the same line followed by the same lowercase are not significantly different (p < 0.05).
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Table 3. Gloss and water contact angle of nano-biocomposite films with different laponite concentrations and for the two surfaces (support and air). Lap Gloss (U.B.) concentration (g/100 g of CS) Support 0 93.8 ± 4.5ª 1.5 87.1 ± 4.1ª 3 87.1 ± 4.1ª 4.5 87.4 ± 5.4ª 6 89.3 ± 5.5a Lap = laponite. CS = cassava starch.
Water contact angle (°) Air 54.1 ± 3.9A,b 45.7 ± 4.6B,b 44.6 ± 4.4B,b 38.5 ± 5.1BC,b 33.5 ± 3.8C,b
Support 75.4 ± 4.6A,a 56.3 ± 7.1B,a 50.1 ± 1.7B,a 46.8 ± 5.7BC,a 42.1 ± 0.2C,a
Air 55.3 ± 0.1A,b 42.7 ± 5.1B,b 29.8 ± 0.7C,b 32.4 ± 2.1C,b 29.9 ± 3.9C,b
All values were expressed as mean ± standard error (n = 3). Means in the same column followed by the same capital letter are not significantly different (p < 0.05). Means in the same line followed by the same lowercase are not significantly different (p < 0.05).
27
