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Incorporation of tetraethylorthosilicate (TEOS) in biodegradable films based on bean starch (Phaseolus vulgaris)
Accepted Manuscript Incorporation of tetraethylorthosilicate (TEOS) in biodegradable films based on bean starch (Phaseolus vulgaris) Karina Oliveira Lima, Bárbara Biduski, Wyller Max Ferreira da Silva, Silvia Moreira Ferreira, Lara Machado Pereira Montenegro, Alvaro Renato Guerra Dias, Daniela Bianchini PII: DOI: Reference:
S0014-3057(16)31182-X http://dx.doi.org/10.1016/j.eurpolymj.2017.02.008 EPJ 7709
To appear in:
European Polymer Journal
Received Date: Revised Date: Accepted Date:
27 September 2016 30 January 2017 6 February 2017
Please cite this article as: Lima, K.O., Biduski, B., da Silva, W.M.F., Ferreira, S.M., Pereira Montenegro, L.M., Dias, A.R.G., Bianchini, D., Incorporation of tetraethylorthosilicate (TEOS) in biodegradable films based on bean starch (Phaseolus vulgaris), European Polymer Journal (2017), doi: http://dx.doi.org/10.1016/j.eurpolymj. 2017.02.008
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Incorporation of tetraethylorthosilicate (TEOS) in biodegradable films based on bean starch (Phaseolus vulgaris)
Karina Oliveira Limaa*, Bárbara Biduskib, Wyller Max Ferreira da Silvab, Silvia Moreira Ferreiraa, Lara Machado Pereira Montenegroa, Alvaro Renato Guerra Diasb, Daniela Bianchinia
a
Centro de Ciências Químicas, Farmacêuticas e de Alimentos, Universidade Federal de
Pelotas, 96010-900 Pelotas, Brasil. E-mail: [email protected], [email protected], [email protected], [email protected] b
Departamento de Ciência e Tecnologia Agroindustrial, Universidade Federal de
Pelotas, 96010-900 Pelotas, Brasil. E-mail: [email protected], [email protected], [email protected]
*Corresponding author: Karina Oliveira Lima ([email protected]), Tel. +00 55 53 98129-6240 (+00 55 53 3279 1471).
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Abstract The starch has been investigated to obtain biodegradable films, but has disadvantages such as hydrophilicity and poor mechanical properties. The addition of inorganic materials can improve mechanical properties by the synergism of the components. The aim of this study was to obtain hybrid films of bean starch and different tetraethylorthosilicate (TEOS) content (5, 20 and 40 g.100 g-1 starch) as well as to evaluate the influence in the properties of films. The films were prepared by casting method with hydrolysis and condensation of TEOS in situ under a controlled pH. The films were characterized after 3 and 15 days of storage, presenting a higher value of Young’s modulus for the film containing 40 g TEOS, lower melting temperature, more hydrophobic surface, and a higher resistance to degradation than starch film. Furthermore, barrier properties improved when stored for 15 days. The relative crystallinity were not affected by adding the precursor.
Keywords: bean starch; TEOS; hybrid films; sol-gel method.
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1. Introduction The effect of pollution generated by the disposal and accumulation of petroleum based plastic on the environment has encouraged research to develop biodegradable packaging obtained from renewable sources [1]. Starch has been investigated for the preparation of biodegradable films, due to its low cost, wide availability, and biodegradability [2, 1]. Starch is a semicrystalline material consisting of two macromolecules, amylose which consists mainly of a straight chain forming an amorphous region in the structure, and amylopectin which gives a crystalline region [3]. The application of starch in the film production is due to the ability of amylose to form a strong and stable network in a solution, with the strong orientation of the chains through hydrogen bonds [4]. The starch bean has 24-65% amylose content, it can vary according to the botanical origin and cultivar. This way, due the high amylose content in bean starch, this grain becomes interesting for developing film [5]. Carioca bean (Phaseolus vulgaris L.) is a Brazilian grains, widely consumed in Brazil, characterized by a light brown tegument with brown stripes [6]. The bean is a legume which has been known as healthy food due to its high contents of protein, dietary fiber and slowly digestible. Because of this, different researchers have studied the physicochemical and processing properties as well as the digestibility of the grains [7, 8] and starch beans [9, 10, 11]. However, the starch of legumes is not as thoroughly studied as the cereals and tubers starches. Therefore, legume starches need to be better studied to facilitate the developments of new applications in both food and non-food products [12, 9]. Starch films may be obtained after a disruption of the granular structure of starch by the gelatinization process, and addition of a plasticizer. The application of native starch for developing films is limited due to its hydrophilic character, which results in a film with both poor mechanical properties and a water barrier when compared to
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synthetic polymers [13, 14, 15, 16, 17]. Thus, the use of additives, such as glycerol (plasticizer), cellulose, and chitosan have been reported to improve the properties of the starch films [18]. Currently there is great interest in the synthesis of hybrid films, consisting of the combination of organic and inorganic components in order to obtain specific properties by the synergism of the components, [19] for instance, the association of the flexibility of organic compounds with improved mechanical properties of an inorganic component. Thus, this enables the formation of a hybrid network. The most common method used for the preparation of hybrid materials is the sol-gel process, which allows control of the chemical composition at mild temperatures by hydrolysis and condensation processes [20]. The precursors usually employed as an inorganic component for the sol-gel process are organoalkoxysilane [20]; among these we highlight tetraethylorthosilicate (TEOS), which may form small particles of silica with a high specific surface area where there are unsaturated chemical bonds. The hydroxyl groups on the surface facilitate the dispersion of macromolecular chains in hybrid films [21]. Some studies about a starch-inorganic hybrid precursor have been carried out [21, 22, 23]. However, relevant studies on water vapor permeability, the mechanical, and thermal characterization of hybrid films, containing only starch and TEOS, and synthesized by sol-gel method, have not been reported in the literature. To our knowledge, this is the first report showing a complete characterization and study of the storage time effect. The aim of this study was to investigate the incorporation of TEOS in biodegradable films based on carioca bean starch on the structural, morphological, thermal, opacity, surface, barrier, and mechanical properties.
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2. Materials and methods 2.1.
Materials Carioca bean grains (Phaseolus vulgaris L.), which were purchased from a local
market (Pelotas, RS), were used for starch isolation. Tetraethylorthosilicate 98% (Si (OC2H5)4) was obtained from Sigma-Aldrich (131903); and all the chemical reagents used in this work had an analytical grade.
2.2.
Isolation of starch The carioca bean starch was isolated according to Rupollo et al. [10], with some
modifications, such as the centrifugation of the starch layer suspended in distilled water at 4000 rpm for 10 min. The brownish top layer was discarded; the underlayer was resuspended in distilled water and recentrifuged at 4000 rpm for 10 min. This process was repeated once more. The resulting material was dried in an oven with air circulation at 40 °C for 16 h until the moisture content was around 9%. The dried starch was milled using basic analytical mill (IKA, A11, USA) and stored at 17 ± 2 °C in a sealed recipient. The starch had approximately 99% purity (0.35% protein, 0.17% lipid and 0.055% ash).
2.3.
Characterization of starch Analysis of the amylose content, morphology, diffraction pattern, thermal analysis
(gelatinization) and nature of the surface of the carioca bean starch was performed such as describe in item 2.4, 2.5, 2.6, 2.7, 2.8, respectively.
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2.4.
Amylose The starch amylose content was determined by a colorimetric method with iodine
according to the method described by McGrane; Rix and Cornell [24]. A suspension with 20 mg of defatted carioca bean starch (d.b.) and 8 mL of dimethylsulfoxide (DMSO) at 90% was stirred and then heated in bath at 85 ºC for 2 h. After cooling, the samples were transferred to a volumetric flask of 25 mL, homogenized and adjusted volume. An aliquot of 1 mL of the solution was added 5 mL of solution of I2/KI (0.0025/0.0065 mol/L) and the volume was completed to 50 mL. The resulting solution was homogenized and allowed to stand for 15 min prior to reading absorbance at 600 nm. To perform the standard amylose curve was used 20 mg of pure potato amylose (Sigma) and realized the same procedure described previous, using aliquots of 0; 0.1; 0.2; 0.4; 0.6; 0.8 and 1.0 mL to determine the absorbance in a spectrophotometer (Jenway, 6705, UK) and construction of the standard curve.
2.5.
Scanning electron microscopy (SEM) The starch morphology was visualized by using a microscope (JEOL, JSM
6610LV, USA). It focused on the sample with an electron beam, with an accelerating voltage of 15 kV. The samples were deposited on copper tape at the aluminum stub. The sample was covered with an Au layer using a Denton Vacuum (Desk V, USA), with the sputtering method for 110 s and 20 mA. The starch Images were performed with a magnification of 500×.
2.6.
X-ray diffraction (XRD) The structural properties of the starch granule and films were determined in the X-
ray diffractometer (Advance Brukers, D8, Germany) using Cu K α (λ = 1.5418 Å) as
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their source. The diffraction angle (2θ) ranged between 5 to 50 °, with a target voltage of 40 kV, a current of 40 mA, a scan increment of 0.0205, and a rate of 1° min-1. The relative crystallinity (RC) was calculated as described by Rabek [25] by the equation RC (%) = (Ac/ (Aa + Ac) * 100), where Ac is the area of crystalline phase, and Aa is the area of the amorphous phase in the X-ray diffractogram.
2.7.
Differential scanning calorimetry (DSC) The gelatinization characteristics of starch were determined by differential
scanning calorimetry (TA-60WS, Shimadzu, Japan) according to the method described by Vanier et al. [11]. Approximately 2.5 mg of starch were weighed in an aluminum pan to which was added distilled water (1:3 w/w). After that the pan was hermetically sealed and allowed to stabilize for 24 h before analysis. The sample was analyzed from 30 to 120 °C, with a heating rate of 10 °C.min-1 in a nitrogen atmosphere. An empty pan was used as a reference. The glass transition temperature, melting temperature, and the enthalpy change were determined for the films. Approximately 10 mg of film were weighed, the sample were analyzed from analyzed from -100 and 250 °C.
2.8.
Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-
FTIR) The nature of the starch and films surface were analyzed by using a Shimadzu spectrometer (IRAffinity-1, Japan), coupled to an Attenuated Total Reflectance accessory (ATR) (Pike Tech, Madison, WI.). The samples were placed on the zinc selenide crystal (ZnSe) and the analysis were performed in the range of 4000 to 500 cm1
, co-adding 32 scans and spectral resolution of 4 cm-1.
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2.9.
Synthesis of the films
Films were prepared by casting method according to Biduski et al. [26], with some modifications. The solutions were prepared with the following proportion: 3.0 g starch:0.3 g glycerol:100 mL distilled water. The solutions were stirred at 300 rpm for 10 min at 90 °C using a thermostatized bath (Quimis, Q214M2). After this time, the filmogenic solution was cooled to 40 °C, followed by a pH adjustment to 9.0 and a slow addition of TEOS at a concentration of 0.0 g, 5.0 g, 20.0 g and 40.0 g TEOS.100 g-1 starch. The temperature was maintained at 40 °C for 1 h and then the solution was homogenized in an ultraturrax (IKA, T18B, Werke, Germany) at 15.500 rpm for 10 min at room temperature. Afterwards, the solution was heated to 40 °C for 1 additional hour. Lastly, 20 g of the solution were spread on an acrylic plate and dried in an oven with forced air circulation (Ethik, Brazil) for 20 h at 30 °C. Before characterization, the films were stored according ASTM D882-12 [27] for 3 and 15 days in a desiccators at 25 °C ± 3 °C with a relative humidity (RH) of 50% ± 3 using a saturated solution of magnesium nitrate hexahydrate (133, VETEC). The RH and temperature were controlled using thermohygrometer. The films were named as ST0, ST5, ST20 and ST40, according to the TEOS concentration.
2.10. Scanning electron microscopy with energy dispersive X-ray spectroscopy (SEMEDS) The surface and cross-section morphology of the films were visualized by a scanning electron microscope (SEM) (JEOL, JSM-6610LV, USA). For the surface analysis, the samples were adhered to the surface of a double sided copper tape at the stub; and for the analysis of the cross section, the films were fractured under liquid N2 and adhered to the r colorless glaze at the stub. The films were covered with a gold layer
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and analyzed by a SEM with an accelerating voltage of 10 kV. The micrographs of the surface and cross section of the films were performed using 1000× and 500× magnification, respectively. An elemental analysis by EDS was performed at different positions on the cross section film.
2.11. Thermogravimetric analysis (TGA) The
degradation temperature
of the
samples
was
obtained
using
a
thermogravimetric analyzer (TA-60WS, Shimadzu, Kyoto, Japan). Samples (4-6 mg) were heated between 30 and 550 °C at a heating rate of 10 °C.min-1 and a nitrogen flow of 50 mL.min-1.
2.12. Opacity of the films The opacity of the films were obtained using a Minolta colorimeter (CR, 400, Japan), and was calculated as the ratio between the value obtained from films overlapping the black standard and the value obtained from the films overlapping the white standard, multiplied by 100 [28].
2.13. Thickness, water solubility and water vapor permeability (WVP) of the films The film thickness was obtained by a digital micrometer (INSIZE, IP-54), according to the ASTM method F2251-13 [29], by the average of eight measurements in random positions for each film. The water solubility was calculated as the percentage of dry matter of the film solubilized after immersion in water at 25 °C for 24 h according to Gontard et al. [30]. The WVP was performed according to the method ASTM E-96/E96M-14 [31] at 25 °C. The samples were sealed with paraffin films on aluminum permeation cells
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(PVA-4, REGMED, Brazil) containing calcium chloride (0% RH). Permeation cells were stored in vacuum desiccators with saturated sodium chloride solution 75% RH. The mass gain of the system was measured for 7 days.
2.14. Mechanical properties of the films The tensile strength, elongation and Young’s modulus of the films were evaluated in a texturometer (TA.TXplus Texture Analyzer), according to ASTM D882-12 [27]. Eight film samples (80 mm × 25 mm) from each film were evaluated. The tensile strength was determined by the maximum stress point from a stress-strain curve. The elongation (strain-specific) was measured as the difference between the distance run until the film ruptured and the initial separation distance (40 mm), multiplied by 100. The Young's modulus was calculated from the inclination of the initial linear region of the stress-strain curve.
2.15. Contact angles Contact angle films were measured using a Drop Shape Analyzer (Kruss) and software- DSA4. The sessile drop method was used with deionized water; a 3 µl droplet of deionized water was gently deposited on the film using a microsyringe and observed with a digital microscope. The values reported are averages of three measurements performed in different areas of sample surface.
2.16. Statistical analysis The analytical determination of the samples was performed in triplicate. The average values and standard errors were demonstrated for each result, except for the relative crystallinity and thermal analysis of the starch and films. At first, the results
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were evaluated by a Q test for rejection of data. After that, the results were compared by Tukey test at a 5% level of significance by an analysis of variance (ANOVA) or a t test to compare the two means, when necessary.
3. Results and discussion 3.1.
Characterization of starch The carioca bean starch showed 34.1% of amylose. According to Singh [5], bean
starch presents a high content of amylose, between 24 to 65%. Therefore, due to its amylose content, the bean starch is an interesting carbohydrate for the development of films. Amylose is a biopolymer with linear chains and when in a solution, it tends to form a strong and stable network through hydrogen bonds between the hydroxyls groups [4]. Fig. 1A shows the morphology of the starch granules of the carioca bean obtained by SEM. The granules have oval and spherical morphologies with the size range between 12 and 36 µm, as well as smooth surfaces without cracks, as reported by Vanier et al.[11]. According to XRD pattern, starches can be classified as A-type (cereals), B-type (tubers), or the mix of both, C-type (leguminous and some seeds). Carioca bean starch presents a C-type diffraction pattern, which is very common in legume plants. Peaks observed in the diffraction angles (2θ) 5.6, 15, 17 and 23 ° (Fig. 1B) are typical for this type of starch. Similar peaks were observed in the study carried out by Rupollo et al. [10] with carioca bean starch stored in different atmospheric conditions. The carioca bean starch used in this study had 34.0% of RC. The characterization of the carioca bean starch by DSC showed that the onset temperature, the peak temperature and the endset temperature of gelatinization were
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66.9, 73.7 and 81.2 ºC, respectively. Besides, the enthalpy change of gelatinization of 9.9 J.g-1 was calculated. Fig. 2 shows the ATR-FTIR spectra of bean starch (Fig. 2A). According to the results shown in Fig. 2A, the spectrum of bean starch has a broad band centered at 3300 cm-1, which is assigned to O-H stretching modes. Bands between 3000 and 2800 cm-1 are assigned to C-H antisymmetric and symmetric stretching modes of methylene groups. Due to the hydrophilic character of the bean starch, the spectrum also shows a band at 1640 cm-1, which is assigned to H-O-H deformation modes of water adsorbed on the surface. The highlighted region between 1160 and 854 cm-1 is assigned to C-O-C vibrational modes of glycosidic bonds of starch [32].
3.2.
Characterization of films
3.2.1. Attenuated total reflectance Fourier transform infrared spectroscopy (ATRFTIR) Fig. 2 shows the ATR-FTIR spectra of the films of bean starch with different TEOS content (Fig. 2B). The spectra of bean starch film (Fig. 2B a) show the same band profile as the spectrum of native bean starch (Fig. 2A). However, the spectra of the films containing the inorganic precursor (Fig 2B b→d) show an enlargement of the bands between 1180 and 850 cm-1 , compared to film ST0, mainly in film ST40 (Fig. 2B d). This suggests the presence of new vibrational modes, asymmetric and symmetric SiO, asymmetric and symmetric Si-O-Si stretching modes of silica obtained from the hydrolysis and condensation of TEOS. He et al. [33] reported similar results. More specifically, 1100–1000, and 950–900 cm-1 was attributable respectively, to ν(Si–O–Si) and ν(Si–OH) [19].
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During the reaction and aging time is expect to occur the condensation of the biopolymer matrix and the inorganic precursor, the broad band assigned to O-H stretching modes between 3500 and 3000 cm-1 suggests a large extension of hydrogen bonds between the components of the film [32].
3.2.2. X-ray Diffraction (XRD) The relative crystallinity of films seems not to be affected by the addition of the precursor TEOS, presenting RC of 10.9, 10.4, 10.0 and 10.5% for ST0, ST5 ST20 and ST40, respectively. It should be noted that RC of native bean starch is 34.0% indicating that the initial crystalline structure was destroyed in the films, after adding glycerol. This new structure was formed during the cooling by recrystallization of amylose in single helices involving glycerol [34]. Peaks in the same 2θ angles, 5.6, 15, 17, 19.5, 22, and 24º, were observed for all films (Fig. 1C). It should be noted that the low ordering degree in the starch films (around 10%) hinders observation of any difference in the crystallinity of the films by XRD analysis. The formation of stable crystalline regions in polymeric materials requires that an economical close packed arrangement of the chains can be achieved in three dimensions, imposes restrictions on the type of chain that can crystallize most readily. Linear and symmetrical chains allow the formation of regular close packing. In addition, chains possessing groups that encourage strong intermolecular attraction thereby stabilizing the alignment of chains [35]. Starch chains have hydroxyl groups that can interact by hydrogen bonds each other and stabilize the structure of biopolymer. It should be reminded that starch is formed by both linear and branched chains, which results in polymeric material with lower degree of crystallinity. The crystallinity of starch films is lower than that observed in the native starch due the addition of
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components with lower molecular weight, such as glycerol. Glycerol also interact by hydrogen bonds and can destroy the interactions among starch chains.
3.2.3. Scanning electron microscopy with energy dispersive X-ray spectroscopy (SEMEDS) Initially, through a visual subjective evaluation all bean starch films had a homogeneous appearance, without blisters. The side of the film in contact with the Petri plate presented a higher brilliance in all cases. The global appearance of the ST5 and ST20 films was not affected by the addition of TEOS. On the other hand, the ST40 film was the most opaque, with different degree of opacity on the same film. The images of the surface and the cross section of films prepared with different TEOS content are shown in Fig. 3 (A, C, E, G) and (B, D, F, H), respectively. The numbers inside the images of the cross section indicates the region where EDS analyses were performed. The surface of films containing the inorganic precursor TEOS is more compact than that observed for the film synthesized without TEOS. Xiong et al. [21] have studied the addition of nano-SiO2 in films of corn starch and polyvinyl acetate. The authors have reported that the addition of nano-SiO2 results in films with smooth and compact surface, suggesting that an increase in the miscibility and compatibility of components had occurred in the film. In addition, the authors explained that nanoparticles of silica have unsaturated bonds on the surface, which favors the hydrogen bonds of silica with hydroxyl groups of starch chains and oxygen atoms of vinyl polyacetate, as well as the formation of Si-O-C bonds. This may explain the compact morphology of the films in this study, suggesting the presence of interactions between starch and an inorganic precursor.
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The EDS analysis, Fig. 3 (B, D, F and H), obtained a semi quantitative analysis of different points in the cross section of the films. If we consider that the organic components of films are richer in C and O atoms, while the inorganic component is richer in Si and O atoms, the EDS analyses can show the distribution of these components in the film. The EDS analyses showed only C and O atoms for the film ST0. On the other hand, the films containing TEOS showed C, O and Si atoms distributed throughout the film. The EDS analyses (Table 2) showed that the distribution of Si was not homogeneous for the films with more than 5% of TEOS. Whereas the other films was observed the Si content is higher in the border than the center for films containing higher TEOS content. The SEM images of surface, Fig. 3 (E and G) showed some concentration of white dots on the surface, which is not observed in the ST5 film (Fig. 3 C). These results suggest the segregation of inorganic material in the border of films due to an excess of the inorganic precursor. A probably saturating Si–OH bonds between starch and silica could be occurred, decreasing the intensity band around 900 cm-1 as observed in the FTIR analysis.
3.2.4. Differential scanning calorimetry (DSC) The melting enthalpy change indicates how much heat is needed in the melting process. Results in Table 1 show that the films containing TEOS have an enthalpy change (∆mH) higher than that obtained for the ST0 film. In this work, the increase of the TEOS content in filmogenic solutions resulted in a decrease of the onset temperature, the peak temperature, and the endset temperature of melting of the films (Table 1). The endothermic peak is associated with the melting of the crystalline phase of starch, which is reorganized during the retrogradation process in the synthesis of film
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[36]. A broadening of peak (Table 1, ∆Tm) was also observed when the TEOS content was increased. The displacement of the peak to lower temperatures, associated with the enlargement of the peak, indicate heterogeneity in the crystalline phase of starch dispersed in the polymeric matrix. In addition, the melting of the crystalline phase occurs in a wide temperature range. It should be noted that the displacement of the melting temperature to lower values and the broadening of the peak are related to the organization of the chains, and consequently, to the size and crystallinity of crystalline phase. The addition of the inorganic precursor seems to affect the organization of chains. It is worth mentioning that the thermogram of the ST0 film showed an abrupt melting at the beginning, with the narrowest peak, suggesting that the crystalline phase is more homogeneous in this film. The Tg is related to the amorphous phase present in the material and indicates a reduction in the movement of the molecular chains with decreasing the temperature analysis. Polymeric materials show a glassy behavior below the Tg. On the other hand, the chains of the polymeric material are flexible above the Tg [37]. According to Frone, Nicolae, Gabor, and Panaitescu [38], certain factors can influence the increase of the characteristic glass transition temperature and melting temperature, such as crosslinking, interactions between chains, and the release of water. The increase in Tg with the addition of TEOS (Table 1, Tg) may be associated with reduced mobility of the polymer chains, which may suggest a strong interaction between the starch chains and the precursor through inter- and intramolecular hydrogen bonds, which gives a greater rigidity for these films. Jia et al. [19] found similar results when studied preparation and properties of poly(vinyl alcohol)/silica nanocomposites derived from copolymerization of vinyl silica nanoparticles and vinyl acetate.
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3.2.5. Thermogravimetric analysis (TGA) The thermal analysis curves shown in Fig. 4 present an initial loss of weight up 120 ºC, which is related to the loss of adsorbed water molecules by the film due to the hydrophilic character. The films ST0, ST5 to ST20 have a similar profile between 120 to 170 °C, the presence of the plateau. This is almost not observed in the film ST40, showing a continuous profile of weight loss. The high silica content in the film, may indicate the slow dehydroxylation of this component during the analysis and possibly a strong interaction with the organic components. Thus, it shows a higher initial resistance to degradation by temperature, as well as a slow and continuous loss. This profile is followed by a weight loss between 170 and 290 °C, which is probably due to the dehydroxylation of the film components associated by hydrogen bonds. According to Lawal et al. [39], the decomposition of glycerol occurs in the range of 120 and 300 °C, which can be occurring in this work. The intense weight loss in only one stage between 290 and 350 °C corresponds to the thermal decomposition of organic matter of the film components. According to Guinesi et al. [40] in this temperature range occur the degradation of starch in nonoxidative conditions, giving mainly CO2, CO, water, acetaldehyde, furan. The ST5, ST20 and ST40 films exhibited a weight loss slightly lower than ST0 film, which is in agreement with the increasing levels of inorganic material added to the films.
3.2.6. Thickness, opacity and mechanical properties of the films The addition of TEOS results in a slight increase in the thickness of the films due to the presence of small particles of silica in the films (Table 3). The ST40 film, stored for 15 days, showed a higher opacity compared to other films (Table 3). However, this
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property was not affected during the storage time of 3 days, which may be related to the aging process [16] as condensation reactions of the TEOS occur during storage [41]. In this storage step condensation reactions between starch and silica species could also occur, giving rise to Si-O-C bonds. However, it is not possible to conclude that this type of chemical bond is present in our films. On the other hand, the opacity can be attributed to a phase separation of the organic and inorganic constituents as observed in the EDS analysis, since optical transparency is a first criterion for the formation of a homogeneous phase [42]. In addition it can be attributed the greater thickness presented, because higher thickness values result in more opaque samples [43] or the insertion of the silica particles in the intermediate spaces of the starch film, preventing the transmission [44]. Table 3 shows a gradual increase of the water solubility at 3 days of storage as the TEOS content is increased, presumably by forming hydrophilic species, such as unsaturated silica particles and small organic molecules containing hydroxyl groups, which are leached from the films during solubility analysis. The water solubility decreased at 15 days of storage, probably due to an improved interaction between the components of the film during the aging process, which convert silanol groups (Si-OH) of silica and alcoholic groups (C-OH) of starch and glycerol in less available species for interactions with water, such as Si-O-Si and Si-O-C. Besides, strong hydrogen bonds between all components of the film could act as crosslinking points between chains and difficult the solubility of films.
3.2.7. Mechanical properties of the films The tensile strength, the elongation percentage, and the Young's modulus of the films with different TEOS content and storage times are shown in Table 3. The higher
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tensile strength and Young's modulus, which are related to the rigidity of the films, were observed for the ST40 film stored for both days (3 and 15) when compared to other TEOS content (ST0, ST5 and ST20), probably due to its greater rigidity. However, the ST20 and ST40 films showed a lower elongation than the ST0 and ST5 films for 15 days of storage, which reinforce that, besides the storage time, the TEOS content affected significantly the mechanical properties of the films. These properties can be correlated with increasing silicon content in the center of the film, as observed by EDS. The inorganic precursor added to the film did not act in the loss of mobility of the chains for 3 days of storage. On the other hand, it is possible to notice a decrease in elongation value for 15 days of storage, suggesting strong interactions of hydrogen bonding between the TEOS, the starch and the glycerol, which can induce a loss of macromolecular mobility and avoid the sliding of a starch chains over the others. This effect was also observed by Lim et al. [14] being reported by the authors an increase in tensile strength and a decrease in elongation percentage of the films, according to the increase of boric acid content. Thus, the authors suggested the formation of a strong intermolecular crosslinking between the two components with covalent bonding, leading to a more rigid structure and hindrance of the sliding of chains [14]. The tensile strength and Young's modulus of the ST0, ST5 to ST20 films increase when different storage times are compared, a fact that occurred in the opposite way in the ST40. This is related probably to the aging process during the storage time, when the rearrangement of starch chains and the condensation of TEOS occur. Thus, films with lower amounts of TEOS become stronger after many days, while the ST40 film seems to be more resistant in a few days. The amount of TEOS in the ST40 film seems to be an excess considering the migration of inorganic component for the
19
borders, which can accelerate the aging process by condensation of TEOS on the surface of the film, as shown by the white dots in the Fig. 3. On the other hand, high amounts of the inorganic component seem to act as reinforcement on mechanical properties of the films. Thus, the silica synthesized in situ during the aging process acts as reinforcement in the polymeric material. Moreover, lower melting temperature of films can be associated to this difficulty in ordering the chains.
3.2.8. Water vapor permeability of the films The water vapor permeability is a measure of the ease with which the water vapor can permeate the material under controlled conditions. According to Fig. 5, films stored for 3 days showed lower values of WVP when compared with those stored for 15 days, except the ST40. ST5 film stored for 3 days showed a decrease in WVP values over 7 days of analyses, the same result was observed for the ST20 film. However, the ST20 film showed a decrease of WVP also for 15 days of storage. The differences in WVP values can be attributed to changes in the film structure during the aging process [45]. Regarding the ST40 film, no significant difference was observed during 7 days of analysis. On the other hand, the storage time seems to be an important parameter to obtain lower WVP values. Films stored for 15 days showed lower WVP values, evidencing that higher concentrations of the inorganic precursor need a longer storage time for the aging process to occur. The water diffusivity in materials and the water solubility coefficient of the film are related directly to the permeability of water vapor in a polymer matrix [46]. Thus, reducing the water transport by diffusion in the polymer, by incorporating a reagent, and decreasing the solubility coefficient, a reduction in values of permeability to water vapor can be achieved. This fact may suggest that, if this
20
material were applied in packaging with a longer shelf life, the interactions between components would reduce the available hydroxyl groups and would decrease the solubility coefficient of the films [47]. The time of storage and addition of inorganic precursor TEOS have an important role to obtain packaging with lower WVP.
3.2.9. Contact angles (CA) The surface hydrophobicity properties of hybrid films were investigated by analysis of the contact angle. The images obtained are illustrated in Fig. 6, the average contact angle of the films were 56.7 ± 0.62d, 58.7 ± 0.05c, 62.3 ± 1.04b, 73.3 ± 0.58a for the ST0, ST5, ST20 and ST40, respectively. It is known that an increase in the contact angle indicates an increase in film hydrophobicity. According to Slavutsky and Bertuzzi [48] the formation of hydrogen bonds between chains reduces the interaction between water and the film surface, which corroborates the increase in hydrogen bonds observed in FTIR results. Thus, a correlation between TEOS content and contact angle can be observed, since the contact angle increases as the inorganic precursor is added. Organic components containing hydroxyl groups and inorganic precursor probably interact by strong hydrogen bonds enough so that act as non-covalent crosslinking points on the chains presenting more hydrophobic surface.
4. Conclusion Homogeneous, bubble-free and transparent hybrid films of carioca bean starch and TEOS were synthesized successfully. The TEOS content and storage time of films develop an important role in the mechanical and barrier properties of the films. The mechanical performance of the hybrid films were improved by adding 40% of the
21
TEOS, which suggests a strong interaction between the components. The water vapor permeability and solubility of films in water were improved when the films were stored for 15 days, allowing the aging process for both starch and the inorganic precursor. Films with a higher TEOS content show more hydrophobic surface, as well as a higher opacity and migration of silica to the border of the film. The migration of silica to the border could avoid the reorganization of the starch chains, and lead to a lower melting temperature for the films.
Acknowledgements We would like to thank CAPES, CNPQ (Ação transversal n° 06/2011 Casadinho/Procad – Process n° 552197/2011-4) and FAPERGS for project financing, CEME-SUL for the microscopy and XRD analyses, and professor Daniel Eduardo Weibel of IQ/UFRGS for the contact angle analyses.
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Table captions Table 1. Thermal properties of bean starch films with different TEOS content. Table 2. Distribution of silicon (Si) throughout the films with different TEOS content. Table 3. Color, opacity and mechanical properties of bean starch films with different TEOS content, stored for 3 and 15 days.
Figure captions Figure 1. Image of carioca bean starch granules (500×) (A), X-ray diffractogram of bean starch (B) and films (C) ST0 (a), ST5 (b), ST20 (c) and ST40 (d) with highlighted peaks.
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Figure 2. ATR-FTIR spectra of bean starch (A) and films (B) ST0 (a), ST5 (b), ST20 (c) and ST40 (d). Figure 3. Images of the surface (A, C, E, G) and the cross section (B, D, F, H) of the ST0 (A, B), ST5 (C, D), ST20 (E, F) and ST40 (G, H) films. Figure 4. Decomposition temperature curves of the ST0 (a), ST5 (b), ST20 (c), and ST40 (d) films. Figure 5. Water vapor permeability (WVP) values of bean starch films with different TEOS contents stored for 3 and 15 days and analyzed during 7 days. The heights of columns represent the mean±desviation values, different letters (a,b,c,d) on the columns indicate significant difference between ST0, ST5, ST20, ST40, and different letters (A,B,C,D,E) on the columns indicate significant difference between the days to same films submitted to the Tukey test (p <0.05). Figure 6. Images of the contact angle (CA) of the ST0 (A), ST5 (B), ST20 (C) and ST40 (D) films.
29
Figure 1.
Figure 2.
30
Figure 3.
31
Figure 4.
Figure 6.
Figure 5. 32
Table 1. Thermal properties of bean starch films with different TEOS content. TEOS
Tm onset
Tm peak
Tm endset
∆Tm (endset- onset) o
∆m H -1
Tg
content (%)
(°C)
(°C)
(°C)
( C)
(J.g )
(°C)
0
165.5
169.0
185.0
19.5
176.3
-30
5
153.3
163.7
183.1
29.8
249.6
15-36
20
113.4
139.0
163.5
50.0
268.4
20-23
40
117.1
133.0
159.4
42.3
223.5
7-20
Tm
onset:
initial crystalline melting temperature; Tm
peak:
peak crystalline melting
temperature; Tm endset: final crystalline melting temperature; ∆Tm: temperature variation; ∆mH: enthalpy; Tg: transition temperature 33
Table 2. Distribution of Si in the films with different TEOS content. Films Upper border Center Bottom border
Si (%)
ST0 -------------
ST5 48.14 55.77 55.78
ST20 76.14 52.80 71.72
ST40 97.65 89.11 97.24
Table 3. Color, opacity and mechanical properties of bean starch films with different TEOS content, stored for 3 and 15 days. Parameters
TEOS content (%)
Storage time (days)
Thickness (mm)
0 5 20 40
3 0.103 ± 0.00b ns 0.106 ± 0.00b* 0.107 ± 0.00b* 0.114 ± 0.00a ns
15 0.099 ± 0.00bc 0.097 ± 0.00c 0.104 ± 0.00b 0.118 ± 0.00a
Opacity
0 5 20 40
10.13 ± 0.32a ns 10.17 ± 0.57a ns 10.43 ± 0.67a ns 10.49 ± 0.27a*
9.98 ± 0.28b 9.33 ± 0.50b 9.46 ± 0.47b 11.81 ± 0.57ª
Water solubility (%)
0 5 20 40
14.35 ± 1.07b* 15.53 ± 0.97ab ns 16.03 ± 0.11ab* 16.53 ± 0.38a*
13.35 ± 1.15ª 14.70 ± 1.05ª 13.83 ± 0.06ª 14.35 ± 0.20ª
Tensile strength (MPa)
0 5 20 40
3.55 ± 0.31b* 3.46 ± 0.48b* 3.39 ± 0.59b* 6.66 ± 0.31ans
5.03 ± 0.35b 4.73 ± 0.40b 4.80 ± 0.28b 6.38 ± 0.31a
Young’s modulus (MPa)
0 5 20 40
48.13 ± 6.85b* 42.77 ± 13.06b* 45.84 ± 12.16b* 226.14 ± 43.70ans
90.29 ± 6.85b 64.57 ± 13.06b 70.87 ± 12.16b 226.76 ± 46.31a 34
0 36.70 ± 0.06ans 40.45 ± 0.08a a* 5 43.47 ± 0.07 32.41 ± 0.05a Elongantion (%) 20 35.90 ±0.14a* 31.83 ±0.14ab 40 30.31 ±0.08a* 20.58 ±0.07b Different lowercase letters in the same column (for each parameter) represent significant differences between the averages submitted to the Tukey test (p <0.05). * and ns are significant and not significant, respectively, for values in the same line submitted to the t test at 5% probability of error.
35
Graphical abstract
36
Highlights Hybrid films with 40% TEOS content significantly increases the Young's modulus Hybrid films with 40% TEOS content presenting higher resistance to degradation Aging time improves the water solubility properties and water vapor permeability Hybrid films showed more hydrophobic surface
37
S0014-3057(16)31182-X http://dx.doi.org/10.1016/j.eurpolymj.2017.02.008 EPJ 7709
To appear in:
European Polymer Journal
Received Date: Revised Date: Accepted Date:
27 September 2016 30 January 2017 6 February 2017
Please cite this article as: Lima, K.O., Biduski, B., da Silva, W.M.F., Ferreira, S.M., Pereira Montenegro, L.M., Dias, A.R.G., Bianchini, D., Incorporation of tetraethylorthosilicate (TEOS) in biodegradable films based on bean starch (Phaseolus vulgaris), European Polymer Journal (2017), doi: http://dx.doi.org/10.1016/j.eurpolymj. 2017.02.008
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.
Incorporation of tetraethylorthosilicate (TEOS) in biodegradable films based on bean starch (Phaseolus vulgaris)
Karina Oliveira Limaa*, Bárbara Biduskib, Wyller Max Ferreira da Silvab, Silvia Moreira Ferreiraa, Lara Machado Pereira Montenegroa, Alvaro Renato Guerra Diasb, Daniela Bianchinia
a
Centro de Ciências Químicas, Farmacêuticas e de Alimentos, Universidade Federal de
Pelotas, 96010-900 Pelotas, Brasil. E-mail: [email protected], [email protected], [email protected], [email protected] b
Departamento de Ciência e Tecnologia Agroindustrial, Universidade Federal de
Pelotas, 96010-900 Pelotas, Brasil. E-mail: [email protected], [email protected], [email protected]
*Corresponding author: Karina Oliveira Lima ([email protected]), Tel. +00 55 53 98129-6240 (+00 55 53 3279 1471).
1
Abstract The starch has been investigated to obtain biodegradable films, but has disadvantages such as hydrophilicity and poor mechanical properties. The addition of inorganic materials can improve mechanical properties by the synergism of the components. The aim of this study was to obtain hybrid films of bean starch and different tetraethylorthosilicate (TEOS) content (5, 20 and 40 g.100 g-1 starch) as well as to evaluate the influence in the properties of films. The films were prepared by casting method with hydrolysis and condensation of TEOS in situ under a controlled pH. The films were characterized after 3 and 15 days of storage, presenting a higher value of Young’s modulus for the film containing 40 g TEOS, lower melting temperature, more hydrophobic surface, and a higher resistance to degradation than starch film. Furthermore, barrier properties improved when stored for 15 days. The relative crystallinity were not affected by adding the precursor.
Keywords: bean starch; TEOS; hybrid films; sol-gel method.
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1. Introduction The effect of pollution generated by the disposal and accumulation of petroleum based plastic on the environment has encouraged research to develop biodegradable packaging obtained from renewable sources [1]. Starch has been investigated for the preparation of biodegradable films, due to its low cost, wide availability, and biodegradability [2, 1]. Starch is a semicrystalline material consisting of two macromolecules, amylose which consists mainly of a straight chain forming an amorphous region in the structure, and amylopectin which gives a crystalline region [3]. The application of starch in the film production is due to the ability of amylose to form a strong and stable network in a solution, with the strong orientation of the chains through hydrogen bonds [4]. The starch bean has 24-65% amylose content, it can vary according to the botanical origin and cultivar. This way, due the high amylose content in bean starch, this grain becomes interesting for developing film [5]. Carioca bean (Phaseolus vulgaris L.) is a Brazilian grains, widely consumed in Brazil, characterized by a light brown tegument with brown stripes [6]. The bean is a legume which has been known as healthy food due to its high contents of protein, dietary fiber and slowly digestible. Because of this, different researchers have studied the physicochemical and processing properties as well as the digestibility of the grains [7, 8] and starch beans [9, 10, 11]. However, the starch of legumes is not as thoroughly studied as the cereals and tubers starches. Therefore, legume starches need to be better studied to facilitate the developments of new applications in both food and non-food products [12, 9]. Starch films may be obtained after a disruption of the granular structure of starch by the gelatinization process, and addition of a plasticizer. The application of native starch for developing films is limited due to its hydrophilic character, which results in a film with both poor mechanical properties and a water barrier when compared to
3
synthetic polymers [13, 14, 15, 16, 17]. Thus, the use of additives, such as glycerol (plasticizer), cellulose, and chitosan have been reported to improve the properties of the starch films [18]. Currently there is great interest in the synthesis of hybrid films, consisting of the combination of organic and inorganic components in order to obtain specific properties by the synergism of the components, [19] for instance, the association of the flexibility of organic compounds with improved mechanical properties of an inorganic component. Thus, this enables the formation of a hybrid network. The most common method used for the preparation of hybrid materials is the sol-gel process, which allows control of the chemical composition at mild temperatures by hydrolysis and condensation processes [20]. The precursors usually employed as an inorganic component for the sol-gel process are organoalkoxysilane [20]; among these we highlight tetraethylorthosilicate (TEOS), which may form small particles of silica with a high specific surface area where there are unsaturated chemical bonds. The hydroxyl groups on the surface facilitate the dispersion of macromolecular chains in hybrid films [21]. Some studies about a starch-inorganic hybrid precursor have been carried out [21, 22, 23]. However, relevant studies on water vapor permeability, the mechanical, and thermal characterization of hybrid films, containing only starch and TEOS, and synthesized by sol-gel method, have not been reported in the literature. To our knowledge, this is the first report showing a complete characterization and study of the storage time effect. The aim of this study was to investigate the incorporation of TEOS in biodegradable films based on carioca bean starch on the structural, morphological, thermal, opacity, surface, barrier, and mechanical properties.
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2. Materials and methods 2.1.
Materials Carioca bean grains (Phaseolus vulgaris L.), which were purchased from a local
market (Pelotas, RS), were used for starch isolation. Tetraethylorthosilicate 98% (Si (OC2H5)4) was obtained from Sigma-Aldrich (131903); and all the chemical reagents used in this work had an analytical grade.
2.2.
Isolation of starch The carioca bean starch was isolated according to Rupollo et al. [10], with some
modifications, such as the centrifugation of the starch layer suspended in distilled water at 4000 rpm for 10 min. The brownish top layer was discarded; the underlayer was resuspended in distilled water and recentrifuged at 4000 rpm for 10 min. This process was repeated once more. The resulting material was dried in an oven with air circulation at 40 °C for 16 h until the moisture content was around 9%. The dried starch was milled using basic analytical mill (IKA, A11, USA) and stored at 17 ± 2 °C in a sealed recipient. The starch had approximately 99% purity (0.35% protein, 0.17% lipid and 0.055% ash).
2.3.
Characterization of starch Analysis of the amylose content, morphology, diffraction pattern, thermal analysis
(gelatinization) and nature of the surface of the carioca bean starch was performed such as describe in item 2.4, 2.5, 2.6, 2.7, 2.8, respectively.
5
2.4.
Amylose The starch amylose content was determined by a colorimetric method with iodine
according to the method described by McGrane; Rix and Cornell [24]. A suspension with 20 mg of defatted carioca bean starch (d.b.) and 8 mL of dimethylsulfoxide (DMSO) at 90% was stirred and then heated in bath at 85 ºC for 2 h. After cooling, the samples were transferred to a volumetric flask of 25 mL, homogenized and adjusted volume. An aliquot of 1 mL of the solution was added 5 mL of solution of I2/KI (0.0025/0.0065 mol/L) and the volume was completed to 50 mL. The resulting solution was homogenized and allowed to stand for 15 min prior to reading absorbance at 600 nm. To perform the standard amylose curve was used 20 mg of pure potato amylose (Sigma) and realized the same procedure described previous, using aliquots of 0; 0.1; 0.2; 0.4; 0.6; 0.8 and 1.0 mL to determine the absorbance in a spectrophotometer (Jenway, 6705, UK) and construction of the standard curve.
2.5.
Scanning electron microscopy (SEM) The starch morphology was visualized by using a microscope (JEOL, JSM
6610LV, USA). It focused on the sample with an electron beam, with an accelerating voltage of 15 kV. The samples were deposited on copper tape at the aluminum stub. The sample was covered with an Au layer using a Denton Vacuum (Desk V, USA), with the sputtering method for 110 s and 20 mA. The starch Images were performed with a magnification of 500×.
2.6.
X-ray diffraction (XRD) The structural properties of the starch granule and films were determined in the X-
ray diffractometer (Advance Brukers, D8, Germany) using Cu K α (λ = 1.5418 Å) as
6
their source. The diffraction angle (2θ) ranged between 5 to 50 °, with a target voltage of 40 kV, a current of 40 mA, a scan increment of 0.0205, and a rate of 1° min-1. The relative crystallinity (RC) was calculated as described by Rabek [25] by the equation RC (%) = (Ac/ (Aa + Ac) * 100), where Ac is the area of crystalline phase, and Aa is the area of the amorphous phase in the X-ray diffractogram.
2.7.
Differential scanning calorimetry (DSC) The gelatinization characteristics of starch were determined by differential
scanning calorimetry (TA-60WS, Shimadzu, Japan) according to the method described by Vanier et al. [11]. Approximately 2.5 mg of starch were weighed in an aluminum pan to which was added distilled water (1:3 w/w). After that the pan was hermetically sealed and allowed to stabilize for 24 h before analysis. The sample was analyzed from 30 to 120 °C, with a heating rate of 10 °C.min-1 in a nitrogen atmosphere. An empty pan was used as a reference. The glass transition temperature, melting temperature, and the enthalpy change were determined for the films. Approximately 10 mg of film were weighed, the sample were analyzed from analyzed from -100 and 250 °C.
2.8.
Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-
FTIR) The nature of the starch and films surface were analyzed by using a Shimadzu spectrometer (IRAffinity-1, Japan), coupled to an Attenuated Total Reflectance accessory (ATR) (Pike Tech, Madison, WI.). The samples were placed on the zinc selenide crystal (ZnSe) and the analysis were performed in the range of 4000 to 500 cm1
, co-adding 32 scans and spectral resolution of 4 cm-1.
7
2.9.
Synthesis of the films
Films were prepared by casting method according to Biduski et al. [26], with some modifications. The solutions were prepared with the following proportion: 3.0 g starch:0.3 g glycerol:100 mL distilled water. The solutions were stirred at 300 rpm for 10 min at 90 °C using a thermostatized bath (Quimis, Q214M2). After this time, the filmogenic solution was cooled to 40 °C, followed by a pH adjustment to 9.0 and a slow addition of TEOS at a concentration of 0.0 g, 5.0 g, 20.0 g and 40.0 g TEOS.100 g-1 starch. The temperature was maintained at 40 °C for 1 h and then the solution was homogenized in an ultraturrax (IKA, T18B, Werke, Germany) at 15.500 rpm for 10 min at room temperature. Afterwards, the solution was heated to 40 °C for 1 additional hour. Lastly, 20 g of the solution were spread on an acrylic plate and dried in an oven with forced air circulation (Ethik, Brazil) for 20 h at 30 °C. Before characterization, the films were stored according ASTM D882-12 [27] for 3 and 15 days in a desiccators at 25 °C ± 3 °C with a relative humidity (RH) of 50% ± 3 using a saturated solution of magnesium nitrate hexahydrate (133, VETEC). The RH and temperature were controlled using thermohygrometer. The films were named as ST0, ST5, ST20 and ST40, according to the TEOS concentration.
2.10. Scanning electron microscopy with energy dispersive X-ray spectroscopy (SEMEDS) The surface and cross-section morphology of the films were visualized by a scanning electron microscope (SEM) (JEOL, JSM-6610LV, USA). For the surface analysis, the samples were adhered to the surface of a double sided copper tape at the stub; and for the analysis of the cross section, the films were fractured under liquid N2 and adhered to the r colorless glaze at the stub. The films were covered with a gold layer
8
and analyzed by a SEM with an accelerating voltage of 10 kV. The micrographs of the surface and cross section of the films were performed using 1000× and 500× magnification, respectively. An elemental analysis by EDS was performed at different positions on the cross section film.
2.11. Thermogravimetric analysis (TGA) The
degradation temperature
of the
samples
was
obtained
using
a
thermogravimetric analyzer (TA-60WS, Shimadzu, Kyoto, Japan). Samples (4-6 mg) were heated between 30 and 550 °C at a heating rate of 10 °C.min-1 and a nitrogen flow of 50 mL.min-1.
2.12. Opacity of the films The opacity of the films were obtained using a Minolta colorimeter (CR, 400, Japan), and was calculated as the ratio between the value obtained from films overlapping the black standard and the value obtained from the films overlapping the white standard, multiplied by 100 [28].
2.13. Thickness, water solubility and water vapor permeability (WVP) of the films The film thickness was obtained by a digital micrometer (INSIZE, IP-54), according to the ASTM method F2251-13 [29], by the average of eight measurements in random positions for each film. The water solubility was calculated as the percentage of dry matter of the film solubilized after immersion in water at 25 °C for 24 h according to Gontard et al. [30]. The WVP was performed according to the method ASTM E-96/E96M-14 [31] at 25 °C. The samples were sealed with paraffin films on aluminum permeation cells
9
(PVA-4, REGMED, Brazil) containing calcium chloride (0% RH). Permeation cells were stored in vacuum desiccators with saturated sodium chloride solution 75% RH. The mass gain of the system was measured for 7 days.
2.14. Mechanical properties of the films The tensile strength, elongation and Young’s modulus of the films were evaluated in a texturometer (TA.TXplus Texture Analyzer), according to ASTM D882-12 [27]. Eight film samples (80 mm × 25 mm) from each film were evaluated. The tensile strength was determined by the maximum stress point from a stress-strain curve. The elongation (strain-specific) was measured as the difference between the distance run until the film ruptured and the initial separation distance (40 mm), multiplied by 100. The Young's modulus was calculated from the inclination of the initial linear region of the stress-strain curve.
2.15. Contact angles Contact angle films were measured using a Drop Shape Analyzer (Kruss) and software- DSA4. The sessile drop method was used with deionized water; a 3 µl droplet of deionized water was gently deposited on the film using a microsyringe and observed with a digital microscope. The values reported are averages of three measurements performed in different areas of sample surface.
2.16. Statistical analysis The analytical determination of the samples was performed in triplicate. The average values and standard errors were demonstrated for each result, except for the relative crystallinity and thermal analysis of the starch and films. At first, the results
10
were evaluated by a Q test for rejection of data. After that, the results were compared by Tukey test at a 5% level of significance by an analysis of variance (ANOVA) or a t test to compare the two means, when necessary.
3. Results and discussion 3.1.
Characterization of starch The carioca bean starch showed 34.1% of amylose. According to Singh [5], bean
starch presents a high content of amylose, between 24 to 65%. Therefore, due to its amylose content, the bean starch is an interesting carbohydrate for the development of films. Amylose is a biopolymer with linear chains and when in a solution, it tends to form a strong and stable network through hydrogen bonds between the hydroxyls groups [4]. Fig. 1A shows the morphology of the starch granules of the carioca bean obtained by SEM. The granules have oval and spherical morphologies with the size range between 12 and 36 µm, as well as smooth surfaces without cracks, as reported by Vanier et al.[11]. According to XRD pattern, starches can be classified as A-type (cereals), B-type (tubers), or the mix of both, C-type (leguminous and some seeds). Carioca bean starch presents a C-type diffraction pattern, which is very common in legume plants. Peaks observed in the diffraction angles (2θ) 5.6, 15, 17 and 23 ° (Fig. 1B) are typical for this type of starch. Similar peaks were observed in the study carried out by Rupollo et al. [10] with carioca bean starch stored in different atmospheric conditions. The carioca bean starch used in this study had 34.0% of RC. The characterization of the carioca bean starch by DSC showed that the onset temperature, the peak temperature and the endset temperature of gelatinization were
11
66.9, 73.7 and 81.2 ºC, respectively. Besides, the enthalpy change of gelatinization of 9.9 J.g-1 was calculated. Fig. 2 shows the ATR-FTIR spectra of bean starch (Fig. 2A). According to the results shown in Fig. 2A, the spectrum of bean starch has a broad band centered at 3300 cm-1, which is assigned to O-H stretching modes. Bands between 3000 and 2800 cm-1 are assigned to C-H antisymmetric and symmetric stretching modes of methylene groups. Due to the hydrophilic character of the bean starch, the spectrum also shows a band at 1640 cm-1, which is assigned to H-O-H deformation modes of water adsorbed on the surface. The highlighted region between 1160 and 854 cm-1 is assigned to C-O-C vibrational modes of glycosidic bonds of starch [32].
3.2.
Characterization of films
3.2.1. Attenuated total reflectance Fourier transform infrared spectroscopy (ATRFTIR) Fig. 2 shows the ATR-FTIR spectra of the films of bean starch with different TEOS content (Fig. 2B). The spectra of bean starch film (Fig. 2B a) show the same band profile as the spectrum of native bean starch (Fig. 2A). However, the spectra of the films containing the inorganic precursor (Fig 2B b→d) show an enlargement of the bands between 1180 and 850 cm-1 , compared to film ST0, mainly in film ST40 (Fig. 2B d). This suggests the presence of new vibrational modes, asymmetric and symmetric SiO, asymmetric and symmetric Si-O-Si stretching modes of silica obtained from the hydrolysis and condensation of TEOS. He et al. [33] reported similar results. More specifically, 1100–1000, and 950–900 cm-1 was attributable respectively, to ν(Si–O–Si) and ν(Si–OH) [19].
12
During the reaction and aging time is expect to occur the condensation of the biopolymer matrix and the inorganic precursor, the broad band assigned to O-H stretching modes between 3500 and 3000 cm-1 suggests a large extension of hydrogen bonds between the components of the film [32].
3.2.2. X-ray Diffraction (XRD) The relative crystallinity of films seems not to be affected by the addition of the precursor TEOS, presenting RC of 10.9, 10.4, 10.0 and 10.5% for ST0, ST5 ST20 and ST40, respectively. It should be noted that RC of native bean starch is 34.0% indicating that the initial crystalline structure was destroyed in the films, after adding glycerol. This new structure was formed during the cooling by recrystallization of amylose in single helices involving glycerol [34]. Peaks in the same 2θ angles, 5.6, 15, 17, 19.5, 22, and 24º, were observed for all films (Fig. 1C). It should be noted that the low ordering degree in the starch films (around 10%) hinders observation of any difference in the crystallinity of the films by XRD analysis. The formation of stable crystalline regions in polymeric materials requires that an economical close packed arrangement of the chains can be achieved in three dimensions, imposes restrictions on the type of chain that can crystallize most readily. Linear and symmetrical chains allow the formation of regular close packing. In addition, chains possessing groups that encourage strong intermolecular attraction thereby stabilizing the alignment of chains [35]. Starch chains have hydroxyl groups that can interact by hydrogen bonds each other and stabilize the structure of biopolymer. It should be reminded that starch is formed by both linear and branched chains, which results in polymeric material with lower degree of crystallinity. The crystallinity of starch films is lower than that observed in the native starch due the addition of
13
components with lower molecular weight, such as glycerol. Glycerol also interact by hydrogen bonds and can destroy the interactions among starch chains.
3.2.3. Scanning electron microscopy with energy dispersive X-ray spectroscopy (SEMEDS) Initially, through a visual subjective evaluation all bean starch films had a homogeneous appearance, without blisters. The side of the film in contact with the Petri plate presented a higher brilliance in all cases. The global appearance of the ST5 and ST20 films was not affected by the addition of TEOS. On the other hand, the ST40 film was the most opaque, with different degree of opacity on the same film. The images of the surface and the cross section of films prepared with different TEOS content are shown in Fig. 3 (A, C, E, G) and (B, D, F, H), respectively. The numbers inside the images of the cross section indicates the region where EDS analyses were performed. The surface of films containing the inorganic precursor TEOS is more compact than that observed for the film synthesized without TEOS. Xiong et al. [21] have studied the addition of nano-SiO2 in films of corn starch and polyvinyl acetate. The authors have reported that the addition of nano-SiO2 results in films with smooth and compact surface, suggesting that an increase in the miscibility and compatibility of components had occurred in the film. In addition, the authors explained that nanoparticles of silica have unsaturated bonds on the surface, which favors the hydrogen bonds of silica with hydroxyl groups of starch chains and oxygen atoms of vinyl polyacetate, as well as the formation of Si-O-C bonds. This may explain the compact morphology of the films in this study, suggesting the presence of interactions between starch and an inorganic precursor.
14
The EDS analysis, Fig. 3 (B, D, F and H), obtained a semi quantitative analysis of different points in the cross section of the films. If we consider that the organic components of films are richer in C and O atoms, while the inorganic component is richer in Si and O atoms, the EDS analyses can show the distribution of these components in the film. The EDS analyses showed only C and O atoms for the film ST0. On the other hand, the films containing TEOS showed C, O and Si atoms distributed throughout the film. The EDS analyses (Table 2) showed that the distribution of Si was not homogeneous for the films with more than 5% of TEOS. Whereas the other films was observed the Si content is higher in the border than the center for films containing higher TEOS content. The SEM images of surface, Fig. 3 (E and G) showed some concentration of white dots on the surface, which is not observed in the ST5 film (Fig. 3 C). These results suggest the segregation of inorganic material in the border of films due to an excess of the inorganic precursor. A probably saturating Si–OH bonds between starch and silica could be occurred, decreasing the intensity band around 900 cm-1 as observed in the FTIR analysis.
3.2.4. Differential scanning calorimetry (DSC) The melting enthalpy change indicates how much heat is needed in the melting process. Results in Table 1 show that the films containing TEOS have an enthalpy change (∆mH) higher than that obtained for the ST0 film. In this work, the increase of the TEOS content in filmogenic solutions resulted in a decrease of the onset temperature, the peak temperature, and the endset temperature of melting of the films (Table 1). The endothermic peak is associated with the melting of the crystalline phase of starch, which is reorganized during the retrogradation process in the synthesis of film
15
[36]. A broadening of peak (Table 1, ∆Tm) was also observed when the TEOS content was increased. The displacement of the peak to lower temperatures, associated with the enlargement of the peak, indicate heterogeneity in the crystalline phase of starch dispersed in the polymeric matrix. In addition, the melting of the crystalline phase occurs in a wide temperature range. It should be noted that the displacement of the melting temperature to lower values and the broadening of the peak are related to the organization of the chains, and consequently, to the size and crystallinity of crystalline phase. The addition of the inorganic precursor seems to affect the organization of chains. It is worth mentioning that the thermogram of the ST0 film showed an abrupt melting at the beginning, with the narrowest peak, suggesting that the crystalline phase is more homogeneous in this film. The Tg is related to the amorphous phase present in the material and indicates a reduction in the movement of the molecular chains with decreasing the temperature analysis. Polymeric materials show a glassy behavior below the Tg. On the other hand, the chains of the polymeric material are flexible above the Tg [37]. According to Frone, Nicolae, Gabor, and Panaitescu [38], certain factors can influence the increase of the characteristic glass transition temperature and melting temperature, such as crosslinking, interactions between chains, and the release of water. The increase in Tg with the addition of TEOS (Table 1, Tg) may be associated with reduced mobility of the polymer chains, which may suggest a strong interaction between the starch chains and the precursor through inter- and intramolecular hydrogen bonds, which gives a greater rigidity for these films. Jia et al. [19] found similar results when studied preparation and properties of poly(vinyl alcohol)/silica nanocomposites derived from copolymerization of vinyl silica nanoparticles and vinyl acetate.
16
3.2.5. Thermogravimetric analysis (TGA) The thermal analysis curves shown in Fig. 4 present an initial loss of weight up 120 ºC, which is related to the loss of adsorbed water molecules by the film due to the hydrophilic character. The films ST0, ST5 to ST20 have a similar profile between 120 to 170 °C, the presence of the plateau. This is almost not observed in the film ST40, showing a continuous profile of weight loss. The high silica content in the film, may indicate the slow dehydroxylation of this component during the analysis and possibly a strong interaction with the organic components. Thus, it shows a higher initial resistance to degradation by temperature, as well as a slow and continuous loss. This profile is followed by a weight loss between 170 and 290 °C, which is probably due to the dehydroxylation of the film components associated by hydrogen bonds. According to Lawal et al. [39], the decomposition of glycerol occurs in the range of 120 and 300 °C, which can be occurring in this work. The intense weight loss in only one stage between 290 and 350 °C corresponds to the thermal decomposition of organic matter of the film components. According to Guinesi et al. [40] in this temperature range occur the degradation of starch in nonoxidative conditions, giving mainly CO2, CO, water, acetaldehyde, furan. The ST5, ST20 and ST40 films exhibited a weight loss slightly lower than ST0 film, which is in agreement with the increasing levels of inorganic material added to the films.
3.2.6. Thickness, opacity and mechanical properties of the films The addition of TEOS results in a slight increase in the thickness of the films due to the presence of small particles of silica in the films (Table 3). The ST40 film, stored for 15 days, showed a higher opacity compared to other films (Table 3). However, this
17
property was not affected during the storage time of 3 days, which may be related to the aging process [16] as condensation reactions of the TEOS occur during storage [41]. In this storage step condensation reactions between starch and silica species could also occur, giving rise to Si-O-C bonds. However, it is not possible to conclude that this type of chemical bond is present in our films. On the other hand, the opacity can be attributed to a phase separation of the organic and inorganic constituents as observed in the EDS analysis, since optical transparency is a first criterion for the formation of a homogeneous phase [42]. In addition it can be attributed the greater thickness presented, because higher thickness values result in more opaque samples [43] or the insertion of the silica particles in the intermediate spaces of the starch film, preventing the transmission [44]. Table 3 shows a gradual increase of the water solubility at 3 days of storage as the TEOS content is increased, presumably by forming hydrophilic species, such as unsaturated silica particles and small organic molecules containing hydroxyl groups, which are leached from the films during solubility analysis. The water solubility decreased at 15 days of storage, probably due to an improved interaction between the components of the film during the aging process, which convert silanol groups (Si-OH) of silica and alcoholic groups (C-OH) of starch and glycerol in less available species for interactions with water, such as Si-O-Si and Si-O-C. Besides, strong hydrogen bonds between all components of the film could act as crosslinking points between chains and difficult the solubility of films.
3.2.7. Mechanical properties of the films The tensile strength, the elongation percentage, and the Young's modulus of the films with different TEOS content and storage times are shown in Table 3. The higher
18
tensile strength and Young's modulus, which are related to the rigidity of the films, were observed for the ST40 film stored for both days (3 and 15) when compared to other TEOS content (ST0, ST5 and ST20), probably due to its greater rigidity. However, the ST20 and ST40 films showed a lower elongation than the ST0 and ST5 films for 15 days of storage, which reinforce that, besides the storage time, the TEOS content affected significantly the mechanical properties of the films. These properties can be correlated with increasing silicon content in the center of the film, as observed by EDS. The inorganic precursor added to the film did not act in the loss of mobility of the chains for 3 days of storage. On the other hand, it is possible to notice a decrease in elongation value for 15 days of storage, suggesting strong interactions of hydrogen bonding between the TEOS, the starch and the glycerol, which can induce a loss of macromolecular mobility and avoid the sliding of a starch chains over the others. This effect was also observed by Lim et al. [14] being reported by the authors an increase in tensile strength and a decrease in elongation percentage of the films, according to the increase of boric acid content. Thus, the authors suggested the formation of a strong intermolecular crosslinking between the two components with covalent bonding, leading to a more rigid structure and hindrance of the sliding of chains [14]. The tensile strength and Young's modulus of the ST0, ST5 to ST20 films increase when different storage times are compared, a fact that occurred in the opposite way in the ST40. This is related probably to the aging process during the storage time, when the rearrangement of starch chains and the condensation of TEOS occur. Thus, films with lower amounts of TEOS become stronger after many days, while the ST40 film seems to be more resistant in a few days. The amount of TEOS in the ST40 film seems to be an excess considering the migration of inorganic component for the
19
borders, which can accelerate the aging process by condensation of TEOS on the surface of the film, as shown by the white dots in the Fig. 3. On the other hand, high amounts of the inorganic component seem to act as reinforcement on mechanical properties of the films. Thus, the silica synthesized in situ during the aging process acts as reinforcement in the polymeric material. Moreover, lower melting temperature of films can be associated to this difficulty in ordering the chains.
3.2.8. Water vapor permeability of the films The water vapor permeability is a measure of the ease with which the water vapor can permeate the material under controlled conditions. According to Fig. 5, films stored for 3 days showed lower values of WVP when compared with those stored for 15 days, except the ST40. ST5 film stored for 3 days showed a decrease in WVP values over 7 days of analyses, the same result was observed for the ST20 film. However, the ST20 film showed a decrease of WVP also for 15 days of storage. The differences in WVP values can be attributed to changes in the film structure during the aging process [45]. Regarding the ST40 film, no significant difference was observed during 7 days of analysis. On the other hand, the storage time seems to be an important parameter to obtain lower WVP values. Films stored for 15 days showed lower WVP values, evidencing that higher concentrations of the inorganic precursor need a longer storage time for the aging process to occur. The water diffusivity in materials and the water solubility coefficient of the film are related directly to the permeability of water vapor in a polymer matrix [46]. Thus, reducing the water transport by diffusion in the polymer, by incorporating a reagent, and decreasing the solubility coefficient, a reduction in values of permeability to water vapor can be achieved. This fact may suggest that, if this
20
material were applied in packaging with a longer shelf life, the interactions between components would reduce the available hydroxyl groups and would decrease the solubility coefficient of the films [47]. The time of storage and addition of inorganic precursor TEOS have an important role to obtain packaging with lower WVP.
3.2.9. Contact angles (CA) The surface hydrophobicity properties of hybrid films were investigated by analysis of the contact angle. The images obtained are illustrated in Fig. 6, the average contact angle of the films were 56.7 ± 0.62d, 58.7 ± 0.05c, 62.3 ± 1.04b, 73.3 ± 0.58a for the ST0, ST5, ST20 and ST40, respectively. It is known that an increase in the contact angle indicates an increase in film hydrophobicity. According to Slavutsky and Bertuzzi [48] the formation of hydrogen bonds between chains reduces the interaction between water and the film surface, which corroborates the increase in hydrogen bonds observed in FTIR results. Thus, a correlation between TEOS content and contact angle can be observed, since the contact angle increases as the inorganic precursor is added. Organic components containing hydroxyl groups and inorganic precursor probably interact by strong hydrogen bonds enough so that act as non-covalent crosslinking points on the chains presenting more hydrophobic surface.
4. Conclusion Homogeneous, bubble-free and transparent hybrid films of carioca bean starch and TEOS were synthesized successfully. The TEOS content and storage time of films develop an important role in the mechanical and barrier properties of the films. The mechanical performance of the hybrid films were improved by adding 40% of the
21
TEOS, which suggests a strong interaction between the components. The water vapor permeability and solubility of films in water were improved when the films were stored for 15 days, allowing the aging process for both starch and the inorganic precursor. Films with a higher TEOS content show more hydrophobic surface, as well as a higher opacity and migration of silica to the border of the film. The migration of silica to the border could avoid the reorganization of the starch chains, and lead to a lower melting temperature for the films.
Acknowledgements We would like to thank CAPES, CNPQ (Ação transversal n° 06/2011 Casadinho/Procad – Process n° 552197/2011-4) and FAPERGS for project financing, CEME-SUL for the microscopy and XRD analyses, and professor Daniel Eduardo Weibel of IQ/UFRGS for the contact angle analyses.
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Table captions Table 1. Thermal properties of bean starch films with different TEOS content. Table 2. Distribution of silicon (Si) throughout the films with different TEOS content. Table 3. Color, opacity and mechanical properties of bean starch films with different TEOS content, stored for 3 and 15 days.
Figure captions Figure 1. Image of carioca bean starch granules (500×) (A), X-ray diffractogram of bean starch (B) and films (C) ST0 (a), ST5 (b), ST20 (c) and ST40 (d) with highlighted peaks.
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Figure 2. ATR-FTIR spectra of bean starch (A) and films (B) ST0 (a), ST5 (b), ST20 (c) and ST40 (d). Figure 3. Images of the surface (A, C, E, G) and the cross section (B, D, F, H) of the ST0 (A, B), ST5 (C, D), ST20 (E, F) and ST40 (G, H) films. Figure 4. Decomposition temperature curves of the ST0 (a), ST5 (b), ST20 (c), and ST40 (d) films. Figure 5. Water vapor permeability (WVP) values of bean starch films with different TEOS contents stored for 3 and 15 days and analyzed during 7 days. The heights of columns represent the mean±desviation values, different letters (a,b,c,d) on the columns indicate significant difference between ST0, ST5, ST20, ST40, and different letters (A,B,C,D,E) on the columns indicate significant difference between the days to same films submitted to the Tukey test (p <0.05). Figure 6. Images of the contact angle (CA) of the ST0 (A), ST5 (B), ST20 (C) and ST40 (D) films.
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Figure 1.
Figure 2.
30
Figure 3.
31
Figure 4.
Figure 6.
Figure 5. 32
Table 1. Thermal properties of bean starch films with different TEOS content. TEOS
Tm onset
Tm peak
Tm endset
∆Tm (endset- onset) o
∆m H -1
Tg
content (%)
(°C)
(°C)
(°C)
( C)
(J.g )
(°C)
0
165.5
169.0
185.0
19.5
176.3
-30
5
153.3
163.7
183.1
29.8
249.6
15-36
20
113.4
139.0
163.5
50.0
268.4
20-23
40
117.1
133.0
159.4
42.3
223.5
7-20
Tm
onset:
initial crystalline melting temperature; Tm
peak:
peak crystalline melting
temperature; Tm endset: final crystalline melting temperature; ∆Tm: temperature variation; ∆mH: enthalpy; Tg: transition temperature 33
Table 2. Distribution of Si in the films with different TEOS content. Films Upper border Center Bottom border
Si (%)
ST0 -------------
ST5 48.14 55.77 55.78
ST20 76.14 52.80 71.72
ST40 97.65 89.11 97.24
Table 3. Color, opacity and mechanical properties of bean starch films with different TEOS content, stored for 3 and 15 days. Parameters
TEOS content (%)
Storage time (days)
Thickness (mm)
0 5 20 40
3 0.103 ± 0.00b ns 0.106 ± 0.00b* 0.107 ± 0.00b* 0.114 ± 0.00a ns
15 0.099 ± 0.00bc 0.097 ± 0.00c 0.104 ± 0.00b 0.118 ± 0.00a
Opacity
0 5 20 40
10.13 ± 0.32a ns 10.17 ± 0.57a ns 10.43 ± 0.67a ns 10.49 ± 0.27a*
9.98 ± 0.28b 9.33 ± 0.50b 9.46 ± 0.47b 11.81 ± 0.57ª
Water solubility (%)
0 5 20 40
14.35 ± 1.07b* 15.53 ± 0.97ab ns 16.03 ± 0.11ab* 16.53 ± 0.38a*
13.35 ± 1.15ª 14.70 ± 1.05ª 13.83 ± 0.06ª 14.35 ± 0.20ª
Tensile strength (MPa)
0 5 20 40
3.55 ± 0.31b* 3.46 ± 0.48b* 3.39 ± 0.59b* 6.66 ± 0.31ans
5.03 ± 0.35b 4.73 ± 0.40b 4.80 ± 0.28b 6.38 ± 0.31a
Young’s modulus (MPa)
0 5 20 40
48.13 ± 6.85b* 42.77 ± 13.06b* 45.84 ± 12.16b* 226.14 ± 43.70ans
90.29 ± 6.85b 64.57 ± 13.06b 70.87 ± 12.16b 226.76 ± 46.31a 34
0 36.70 ± 0.06ans 40.45 ± 0.08a a* 5 43.47 ± 0.07 32.41 ± 0.05a Elongantion (%) 20 35.90 ±0.14a* 31.83 ±0.14ab 40 30.31 ±0.08a* 20.58 ±0.07b Different lowercase letters in the same column (for each parameter) represent significant differences between the averages submitted to the Tukey test (p <0.05). * and ns are significant and not significant, respectively, for values in the same line submitted to the t test at 5% probability of error.
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Graphical abstract
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Highlights Hybrid films with 40% TEOS content significantly increases the Young's modulus Hybrid films with 40% TEOS content presenting higher resistance to degradation Aging time improves the water solubility properties and water vapor permeability Hybrid films showed more hydrophobic surface
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