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Cellulose fiber reinforced biodegradable films based on proteins extracted from castor bean (Ricinus communis L.) cake
Industrial Crops and Products 67 (2015) 355–363
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Cellulose fiber reinforced biodegradable films based on proteins extracted from castor bean (Ricinus communis L.) cake T.G. Oliveira, G.L.A. Makishi, H.N.M. Chambi, A.M.Q.B. Bittante, R.V. Lourenc¸o, P.J.A Sobral ∗ Department of Food Engineering – FZEA/USP, Av. Duque de Caxias Norte, 225, 13635-900 Pirassununga, SP, Brazil
a r t i c l e
i n f o
Article history: Received 17 September 2014 Received in revised form 17 January 2015 Accepted 19 January 2015 Keywords: Biopolymers Composite Thin material Mechanical properties Microstructure
a b s t r a c t The aim of this study was to develop films based on proteins extracted from castor bean (Ricinus communis L.) cake, reinforced with cellulose fibers for use in agriculture, as bags for planting seedlings. The specific aims was to study the effect of fibers concentration on the mechanical properties, color, opacity, gloss, moisture, solubility in water, water vapor permeability (WVP), microstructure, thermal properties, and chemical structure through Fourier transform infrared spectroscopy (FTIR). Proteins were extracted from castor bean cake in a reactor and then freeze-dried. The cellulose fibers were dispersed in water using a high-speed stirrer. The films were produced by dehydration of film-forming solutions (FFS) prepared with the freeze-dried protein (6 g/100 g FFS), cross linker (5 g glyoxal/100 g protein), plasticizer (30 g glycerol/100 g protein), and fibers (0; 2.5; 5; 7.5; 10; and 12.5 g cellulose fibers/100 g protein). The fiber addition had no effect on thickness, humidity, solubility in water and water vapor permeability of the films. In contrast, an increase in puncture force, tensile strength and elastic modulus, and a decrease in the elongation at break were observed as a function of fiber concentration. The fiber addition also affected color, opacity and gloss of the films. Scanning electron microscopy analysis showed that the cellulose fibers were well dispersed in the film matrix, explaining its effect on the mechanical properties of the films. The analysis by Fourier transform infrared spectroscopy (FTIR) corroborated these results. The main conclusion of this study is that the load of cellulose fibers improved the mechanical properties of films produced with the freeze-dried castor bean cake protein. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Environmental concerns associated with plastic waste management have emphasized the importance of developing biodegradable materials to reduce the problems of disposal of plastic waste (Fang et al., 2005). A potential material for the production of biodegradable plastics is the films based on biopolymers such as proteins and polysaccharides, which besides their biodegradable nature, are from renewable resources (Sobral, 2000). Fibers are used as reinforcement materials in biodegradable films to reduce costs and improve it mechanical properties (Kunanopparat et al., 2008; Salgado et al., 2008). Several studies on composites reinforced with vegetable fibers can be found in literature, including wheat gluten based films reinforced with hemp (Kunanopparat et al., 2008), wheat flour based films reinforced with cotton fiber (Dobircau et al., 2009), wheat gluten and fiber
∗ Corresponding author. Tel.: +55 19 35654192; fax: +55 19 35654114. E-mail address: [email protected] (P.J.A Sobral). http://dx.doi.org/10.1016/j.indcrop.2015.01.036 0926-6690/© 2015 Elsevier B.V. All rights reserved.
˜ wheat straw based films (Montano-Leyva et al., 2013), corn starch based films reinforced with keratin, lignin and firs cellulose fibers (Bodirlau et al., 2013), composites of soluble potato starch or corn starch reinforced with sugarcane fibers (Gilfillan et al., 2012), cassava starch based films reinforced with wheat straw fibers (Famá et al., 2009), flax-fiber reinforced PLA biocomposites films (Arias et al., 2012), among others. Among the vegetable fibers, the cellulose fiber is most privileged in studies on biopolymer based films. A highly purified wood pulp composed of 92–98% cellulose, known as dissolved pulp was used in the manufacture of cellulose-derived products (Wertz et al., 2010), and is the most common raw material of microfibrils cellulose (MFC) that can be isolated by employing well-known mechanical treatment methods such as homogenization, microfluidization, microgrinding, and cryocrushing. The stability of its structure confers low solubility of cellulose in almost all reagents and great mechanical strength to the microfibrils (Agoda-Tandjawa et al., 2010; Rezayati Charani et al., 2013; Silva et al., 2008). The cellulose fiber has been applied to produce composites of polyurethane resin derived from castor bean oil (Miléo et al., 2011), starch based
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films (Müller et al., 2009), composite films from galactoglucomannans (Mikkonen et al., 2011) and rice flour based films (Dias et al., 2011). The castor bean (Ricinus communis L.) is an oilseed with high economic value because it presents a well-defined market for the oil. Currently, great interest has been given to castor bean cake generated during oil extraction process for its high protein content, which can reach 40% (Lacerda et al., 2014; Silva et al., 2012; Visser et al., 2011). The castor bean cake may be a viable alternative for the development of biodegradable films, contributing to the success of this biodiesel agribusiness (Makishi et al., 2013; Bittante et al., 2014). But considering that this protein was obtained by solubilization, the major peptide fractions was around 40 and 20 kDa (Lacerda et al., 2014). Then, it could be interesting some degree of modification for increase it molecular weight to guarantee a good film-forming property. Chambi et al. (2014) modified that proteins using tannic acid and produced very workable films. And Makishi et al. (2013) produced films of similar proteins cross linked with glutaraldehyde and glyoxal and observed that glyoxal implied in films with improved mechanical properties and lower film solubility than glutaraldehyde. These behaviors can be explained by the high content of arginine in the protein, which react with glyoxal (Makishi et al., 2013). These materials could have applications in agriculture, particularly as bags for planting seedlings (Bittante et al., 2014). Conventionally, these bags are produced using low-density polyethylene (LDPE) films, which have good physical properties, principally it high mechanical resistance (tensile strength = 34 MPa) (Chambi et al., 2014). However, it is well known that the LDPE is not biodegradable and its bags are difficult to recycle due to the large amount of organic matter adhered to material. The aim of this paper was to develop films based on proteins extracted from the castor bean (R. communis L.) cake reinforced with cellulose fibers interesting for using as bags for plants seedling. Specifically, it was studied the effect of fiber concentration on the mechanical properties (puncture and tensile tests), color, opacity, gloss, humidity, solubility in water, water vapor permeability, microstructure, thermal properties, and chemical structure through Fourier transform infrared spectroscopy. 2. Material and methods Castor bean cake (CBC) was supplied by Azevedo Óleos Indústria e Comercio de Óleos Ltda (Itupeva-SP). Sodium hydroxide and glycerol were purchase from Synth and glyoxal from Sigma. Cellulose fibers (CF) are conventional bleached eucalyptus kraft pulp from Votorantim Cellulose and Paper, Brazil provided from Construction and Environment Group, Faculty of Animal Science and Food Engineering (Savastano et al., 2009). Similar fibers were used to produce starch-based composite films (Dias et al., 2011). 2.1. Proteins extraction from castor bean cake Extractions of the protein from CBC were performed in a 4.5 L reactor (Bio-Tec-V, TECNAL) with controlled temperature (50 ◦ C), pH (12) and agitation (400 rpm) (Lacerda et al., 2014; Makishi et al., 2013). The freeze-dried extracts were analyzed for protein determination by the classical Kjeldahl method (A.O.A.C, 1995). More data on freeze-drying conditions and on the composition of the extracted freeze-dried proteins can be found in a paper published previously (Makishi et al., 2014). 2.2. Cellulose fibers dispersion Cellulosic pulp was dispersed in water by mechanical stirring (3000 rpm for 5 min) (Mármol et al., 2013). The excess water was
removed by manual clamping filtration producing the CF. The humidity of this dispersed pulp was determined by oven drying at 105 ◦ C for 24 h. 2.3. Films preparation The films were produced by casting process at room temperature (22 ± 1 ◦ C). Film-forming solutions (FFS) were prepared with five different CF concentrations (0; 2.5; 5; 7.5; 10 and 12.5 g cellulose fibers/100 g protein). For this preparation, firstly, the CF were dispersed in distilled water under continuous stirring (400 rpm) for 5 min, and then, the freeze-dried Castor bean protein (FDCBP) was slowly added into this CF dispersion under continuous stirring for more 30 min. After FDCBP dispersion, the cross linker (5 g glyoxal/100 g of protein) was added. After 5 min, the plasticizer was also added (30 g glycerol/100 g protein) always under constant magnetic stirring, for 1 min. The FFS were applied to Petri dishes (150 × 15 mm) (Bioplass) and dried in an oven with forced air circulation (Marconi, MA037) at 30 ◦ C and controlled relative humidity (55–65%), for 16 h (Sobral et al., 2001). Before characterizations, the films were conditioned in desiccators containing saturated solutions of Mg(NO2 )2 (52.5% relative humidity) at 25 ◦ C for at least 7 days, except for the samples for scanning electron microcopy and atomic force microscopy analyses, which were conditioned in desiccators with NaOH and in silica gel, respectively, for 7 days. 2.4. Characterization of the cellulose fibers CF were characterized by scanning electron microscopy (SEM), (Hitachi, TM3000; Tokyo, Japan), with a tungsten electron source at 10 kV. Aliquots of a CF solution (1 g CF/50 g of water) were deposited directly onto the carbon tape and dried for 24 h in an oven. 2.5. Characterization of the films All characterizations were carried out in triplicate, at least. 2.5.1. Films thickness The film thickness was determined using a digital micrometer (0.001 mm; Mitutoyo) with a 6.4 mm diameter probe, averaging ten different positions (Sobral et al., 2001). 2.5.2. Humidity and solubility in water The humidity of the films, expressed as g of water/100 g of wet material, was determined by oven drying at 105 ◦ C for 24 h (Gontard et al., 1994). And the solubility in water of the films was determined at 25 ◦ C by immersing the films (d ∼ 2 cm) in distilled water (50 ml) under slight stirring with the aid of a shaker (Marconi, MA141) for 24 h, according to Gontard et al. (1994). The material was filtered through filter paper (Nalgon), weighed and then dried in an oven at 100 ◦ C for 24 h for determination of dry weight. Considering that the initial dry mass of the samples was known, the solubility of the films was calculated as dissolved dry mass. 2.5.3. Mechanical properties The mechanical properties of the films were determined by tensile (tensile strength, elongation at break and elastic modulus) and puncture (puncture force and deformation) tests, using a texturometer TA.XT2i (TA instruments, Surrey, UK), at room temperature (22–25 ◦ C). For the tensile tests, film samples were cut into rectangles (15 mm × 100 mm) and fixed on the grips, separated by 50 mm. These samples were submitted to traction at 1 mm/s (Thomazine et al., 2005). For puncture tests, the films were cut into 46 mm-diameter circles, fixed on a cell, and submitted to perforation by a cylindrical
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probe (d = 3 mm), moving at 1 mm/s (Sobral et al., 2001). The force and displacement at puncture were measured. Puncture deformation (PD, %) was calculated from the distance traveled by the probe to the puncture point, using Eq. (1).
PD(%) = [
(d2 + l02 ) − l0 l0
] × 100
(1)
where l0 is the cell radius (23 mm) and PD: Puncture deformation (%). 2.5.4. Water vapor permeability The water vapor permeability (WVP) was determined gravimetrically, at 25 ◦ C, according to Thomazine et al. (2005). The samples were fixed on aluminum cell containing silica gel (0% RH) and placed in a desiccator containing distilled water (100% RH). These cells were weighed (±0.01 g) daily for 7 days to guarantee the steady state permeation, using a semi-analytical balance (Marte, AS2000, Sao Paulo, Brazil). 2.5.5. Color and opacity The color of the films was determined with a colorimeter Miniscan XE (HunterLab) working with D65 (daylight) lamp, at 10◦ , and measuring with an opening of 30 mm. For color determination, the films were placed on the surface of the standard white plate and de parameters L*, a*, and b* were measured (Gennadios et al., 1996; Kunte et al., 1997). The total color difference (E*) was calculated using Eq. (2): E ∗ =
L∗2 + a∗2 + b∗2
(2)
where L* = L*sample – L*standard (92.58 ± 0.92); a* = a* sample – a* standard (−0.88 ± 0.06); b* = b* sample – b* standard (1.26 ± 0.02). The opacity was measured using the same equipment and computer program used in color measurements. Film opacity was determined according to the Hunterlab method, in the reflectance mode (Sobral, 2000). Opacity (Y) was calculated from the relationship (Y = Yb/Yw) between the opacity of the film superimposed on the black standard (Yb), and that of the film superimposed on the white standard (Yw). 2.5.6. Gloss The gloss of the film surfaces was determined using the Rhopoint NGL 20/60 glossmeter, at angle of 20◦ (Alves et al., 2011; Moraes et al., 2008). The measurements were performed on both surfaces of the films.
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(Carvalho and Grosso, 2004). Both drying surfaces and that in contact with the support were analyzed. The used SEM was coupled to an EDS (energy dispersive spectroscopy detector).
2.5.8. Atomic force microscopy (AFM) These analyses were carried out using an atomic force microscope (model NT-MDT Solver Next brand), equipped with a software for images analysis (New Model 3.1.0 program PX). The films were cut into areas of 2 × 2 cm for use in the AFM. The equipment was operated in tapping mode using NSG tip. The images were recorded using scanning rates of 0.5 Hz. The nominal spring constant and nominal resonant frequency was 11.8 N/m and 240 kHz, respectively.
2.5.9. Fourier transform infrared spectroscopy (FTIR) Fourier transform infrared spectroscopy (FTIR) spectra were recorded using a PerkinElmer spectrometer Spectrum One (PerkinElmer one) equipped with accessory UATR, according to Vicentini et al. (2005). The spectra were recorded in the range of 4000–400 cm−1 with a resolution of 2 cm−1 . Data were analyzed using the Spectrum One (Spectrum One version 5.3) software.
2.5.10. Thermal properties The films thermal properties determination was accomplished by differential scanning calorimetry using a DSC TA 2010 controlled by a TA 5000 module (TA instruments, USA) with quench cooling accessory (Sobral et al., 2001). Aliquots were weighed (∼10 mg) in a precision balance (±0.0001 g) (Scientech, SA210) and placed in hermetic aluminum pans. The system was heated at a rate of 5 ◦ C/min, between −150 and 150 ◦ C in an inert atmosphere (45 ml/min N2 ). The reference was a empty pan. The glass transition temperature (Tg ) was calculated as the inflexion point of the baseline, caused by the discontinuity of the specific heat of the sample. The melting temperature (Tm ) was calculated where the endothermic peak occurred. The enthalpy (Hm) of the sol–gel transition was determined from the endothermic peak area. The properties were calculated with the help of V1.7F Universal Analysis (TA Instruments) software (Sobral et al., 2001).
1.2. Statistical analysis 2.5.7. Scanning electron microscopy The microstructure of films were analyzed using a scanning electron microscope (SEM), (Hitachi TM3000; Tokyo, Japan) with a tungsten electron source at 5 kV, without previous treatment
The results were analyzed using ANOVA and Tukey’s mean comparison test (p ≤ 0.05) was performed using the statistical program “Statistical Analysis Systems” – SAS.
Fig. 1. Scanning electron micrographs of cellulose fibers: (A) cellulose pulp fiber [800× magnification] and, (B) dispersed cellulose fiber [1200× magnification].
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Fig. 2. Effect of cellulose fiber concentration (%) on total difference of color (E*) of freeze-dried castor bean protein based films (– calculated by the Eq. (3)), and on gloss at 20◦ ( side in contact with the plate, side in contact with the air; – calculated by the Eq. (4) and, – and by the Eq. (5). Different letters show significant difference for the same parameters.
3. Results and discussion
composites with pulp cellulose fibers (∼15%) by Curvelo et al. (2001). The solubility in water of the FDCBP based films reinforced with varied from 35.9 to 41.4%, with no significant difference (p > 0.05) among treatments, having an average of 39.33 ± 1.89%. Both the control films and those containing CF maintained their integrity even after 24 h of water immersion, without separation between the matrix and fibers, showing that the intermolecular networks remained unchanged and only low molecular peptides and plasticizer could have solubilized (Wittaya, 2009). This behavior is important for the biodegradation process in soil. Bags based on LPDE are not soluble in water.
3.1. Characterization of fibers The CF presented oval shape and small pores on its surface, formed by grouped fibrils (Fig. 1). The diameters of the CF ranged from 8 to 12 m. Similars results can be found in the work of Chen et al. (2011) and Lu et al. (2014). Savastano et al. (2009) worked with similar cellulose fibers from Eucaliptus grandi and observed fiber length of 0.66 mm and an aspect ratio of 61. They also found a kappa number, what is an indication of residual lignin content in chemical pulps, of 6.1 that indicates the presence of a small content of lignin in the surface of cellulose fibers. According to Dias et al. (2011), the hydroxyl groups of lignin can interact with glycerol, acting as an interfacial compatibilizer between fibers and the matrix, leading to films with higher tensile strength.
3.2.3. Water vapor permeability (WVP) The CF addition in the FDCBP matrix did not statistically affect (p > 0.05) the WVP of films, which remained around 0.73 ± 0.06 g mm/h m2 kPa. Quite possibly, the presence of pores, as discussed in the microscopy results, may have contributed to this behavior. Similar behavior was observed in cassava starch based films reinforced with cellulose fibers, but this behavior was explained by the small variation in water solubility inside films and diffusion coefficient of the films (Müller et al., 2009). Unfortunately, the load with cellulose was not able to transform the material in a high water vapor barrier material, as the LPDE. Nevertheless, this is not necessarily a limitation property for application as bags for plating seedling.
3.2. Characterization of films 3.2.1. Visual aspects and thickness The films were easily removed from the Petri dishes, and had good flexibility and were ease of cutting regardless of the concentrations of CF. Films without CF were brighter than the films containing CF, once they had a smoother texture than the other films. Among the films with CF, visual differences were not observed as a function of the fiber concentration. The presence of CF had no significant (p > 0.05) effect in the film thickness, which remained around a mean value of 0.127 ± 0.004 mm. The films thickness was higher than films based on LPDE for bags for plant sendling, which usually remain around 0.060 mm (Chambi et al., 2014).
3.2.4. Color and opacity The color parameters were measured only on the drying surface of the films, i.e., on the upper surface. The mean L*, a* and b* values were 15.47 ± 1.63; 26.29 ± 1.47; and 19.35 ± 1.24, respectively, with no significant difference (p > 0.05) among treatments. Such values were considered high for films, and imply dark materials that could be interesting to use in agricultural domain. On another hand, the total color difference (E*), which expresses the color of the material, decreased linearly (Eq. (3)) as a function of the CF concentration (Fig. 2). The color parameters of the FDCBP used as raw material were high (L* = 52.42 ± 0.01,
3.2.2. Humidity and solubility in water The addition of CF did not affect (p > 0.05) the films humidity, which had an average value of 14.35 ± 0.57%, allowing to suggest that CF did not cause changes in the films hygroscopicity. The humidity values were close to those found in corn starch based
Table 1 Elastic modulus, puncture deformation and opacity of freeze-dried castor bean protein films reinforced with cellulose fibers as a function of cellulose concentration (Cfibers ). Cfibers (g/100 g protein) 0 Elastic modulus(MPa/%) Puncture deformation(%) Opacity
2.5
1.43 ± 0.18 7.50 ± 0.26a 15.91 ± 1.02b
b,c
5
1.32 ± 0.38 6.87 ± 0.45a,b 28.56 ± 12.69a,b c
7.5
1.80 ± 0.32 7.24 ± 0.54b,c 30.91 ± 7.9a,b
Different lowercase letters in the same line show significant difference for the same parameter.
a,b
10
1.82 ± 0.29 6.65 ± 0.33c 29.94 ± 9.67a,b a,b
12.5
2.05 ± 0.48 6.66 ± 0.26c 33.65 ± 7.49a,b a
1.70 ± 0.40a,b,c 6.58 ± 0.36c 45.31 ± 12.33a
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Fig. 3. Effect of cellulose fiber concentration (%) on tensile strength and elongation at break of freeze-dried castor bean protein based films (– calculated by the Eq. (6) and, – by the Eq. (7)), and on puncture force of freeze-dried castor bean protein based films (– calculated by the Eq. (8). Different letters show significant difference for the same parameter.
a* = 6.05 ± 0.04, b* = 18.44 ± 0.03) because it is a dark material, due the presence of pigments as quinone (Lacerda et al., 2014), in contrast to cellulose fibers, which, being white, presented low values (L* = 94.02 ± 2.05, a* = 0.05 ± 0.01, b* = 4.27 ± 0.05). Thus, the
decrease of the total color difference of the FDCBP based films must have been due to the mixture of both products. E ∗ = −0.35Cfiber + 86.5(R2 = 0.9704)
(3)
Fig. 4. Scanning electron micrographs of drying surface (A1 and B1) and of support contact surface (A2 and B2) of freeze-dried castor bean cake protein based films reinforced with cellulose fibers at (A1 and A2) 0 and (B1 and B2) 12.5 g of fiber/100 g protein. 500× magnification.
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Fig. 5. Micrographs of a salt crystal in freeze-dried castor bean cake protein based films obtained by (A) scanning electron microscopy and (B) atomic force microscopy.
where E*:Total color difference and Cfiber : Fiber concentration (%). The opacity of the films varied (p < 0.05) with the CF concentration, ranging from 15.9 to 45.3 when the CF concentration increased from zero to 12.5% (Table 1). These values can be considered a high value for protein films, which means that these films can be considered almost as opaque ones. Usually, bags for planting seedling are dark and opaque, to avoid the sun penetration in soil. But in despite of the reduction in the total color difference provoked by the cellulose load, this material can be considered as dark (E* > 82), and thus, having potential to be used as bags for plating seedling. The effect of cellulose load was positive and corroborate with this last statement. 3.2.5. Gloss The gloss of the films surfaces was determined at an angle of 20◦ . For all films, a decrease in gloss was observed in both the drying surface and on the surface in contact with the support, which was dependent of CF concentration (Fig. 2). In the drying surface, the gloss values decreased linearly (Eq. (4)) from 9.4 to 0.4 when the CF concentration increased from zero to 12.5%, while for support contact surface this decrease was (Eq. (5)) from 14.7 to 3.4. No data on gloss of bags based on LPDE was found. Drying surface : G = −0.69Cfiber + 8.4(R2 = 0.9547)
(4)
Contact surface : G = −0.86Cfiber + 14.0(R2 = 0.9544)
(5)
where G: Gloss
(20◦ )
and Cfiber : Fiber concentration (%).
3.2.6. Mechanical properties 3.2.6.1. Tensile tests. The addition of CF in the film matrix provoked a linear increase (Eq. (6)) in tensile strength as a function of CF concentration, evidencing a strong positively influence of the CF on the mechanical properties of the composites (Fig. 3). The maximum CF concentration resulted in an increase of approximately 42% in the tensile strength of the composite films as compared to control, suggesting that the FDCBP based composites reinforced with cellulose fiber presented adequate dispersion of fiber in the biopolymer matrix (Miléo et al., 2011). Nevertheless, the improved material (TS = 13 MPa) was always less resistant then the LPDE-based bags. But in spite of that, the FDCBP based composite could be indicated to bags for planting with need low quantity of soil. TS = 0.35Cf iber + 8.4(R2 = 0.865)
(6)
where TS: Tensile strength (MPa) and Cfiber : Fiber concentration (%). Dobircau et al. (2009) observed a similar behavior working on rice flour starch based films, where the increase in tensile strength
was attributed to good dispersion of the fiber in biopolymer matrix, demonstrating that the stress transfers from the fiber to the matrix occurred properly. Dias et al. (2011) studied the effect of the addition of cellulose fibers on rice flour based films, and found that the addition of 0.3 g fibers/g dried flour (plasticized with 0.3 g glycerol/g flour) improved the mechanical properties of the films. Similar results were obtained by Miléo et al. (2011), who studied castor oil composites reinforced with sugarcane straw cellulose fibers at various concentrations (0, 5, 10, 15 and 20% w/w), and also observed an increase in the tensile strength. Regarding the elongation at break, it was noted that this mechanical property decreased exponentially (Eq. (7)) with increasing of CF concentration in FDCBP based films (Fig. 3), suggesting that the strong protein/fibers interactions restricted the movement of protein chains identical to an anti-plasticizing behavior. Increasing the fiber concentration in the films resulted in a decrease in the elongation at break from 110.67 to 23.26%, as compared to the films with 12.5% cellulose fiber in relation to the control. The observed decreasing in FDCBP based films EB do not constitute a limitation for use as bag for planting seedling. EB = 92.5e−0.12Cf iber (R2 = 0.8963)
(7)
where EB: Elongation at break (%) and Cfiber : Fiber concentration (%). The decrease in the elongation at break as a function of increasing CF concentration was also observed in rice flour based films reinforced with cellulose fibers plasticized with 0.3 g glycerol/g flour, with values from 8.8 to 66.4% when 0.3 g fibers/g flour was added (Dias et al., 2011). In cassava starch based films reinforced with cellulose fibers, the elongation at break decreased by up to 18 times in films with 0.5 g fibers/g starch when compared with the pure matrix (Müller et al., 2009). The addition of CF as reinforcement in FDCBP based films resulted in an increase in the elastic modulus of the films (Table 1), without necessarily follow a monotonic behavior. The maximum value for this module was observed for films with 10% in CF, representing an increase of 30.2% as compared to the control film. This behavior is in agreement with studies on polyurethane from castor oil films reinforced with cellulose fiber from sugarcane bagasse (Miléo et al., 2011). 3.2.6.2. Puncture tests. The results of the mechanical properties determined by puncture tests corroborated with the results of tensile strength. The puncture force showed a linear increase (Eq. (8)) as a function of CF concentration (Fig. 3). PF = 0.38Cfiber + 10.7(R2 = 0.9704)
(8)
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Fig. 6. Scanning electron micrographs of cryofractured surfaces of freeze-dried castor bean cake protein based films reinforced with cellulose fibers at (A) 0 and (B) 12.5 g of fiber/100 g protein. 1200× magnification.
where PF: Puncture force (N) and Cfiber : Fiber concentration (%). Similar to the results for tensile tests, the puncture deformation decreased with increasing CF concentration (Table 1), although this decrease was less pronounced than in the tensile test. 3.2.7. Microstructure of the films The scanning electron micrographs of films surfaces (Fig. 4) presented circular microstructures, not visible to the naked eye, and in greater amounts in the drying surface, independent of the CF concentration. These particles were agglomerates of salts, possibly formed from the NaOH used in the protein extraction (Lacerda et al., 2014), that was dragged during drying, thus explaining the highest concentration in the drying surface. And apparently, the amount of these particles in the surface increased with CF concentration in films possible due to the increasing of some porosity in films. Fig. 5 shows these salt crystals more clearly being that AFM microscopy showed that these crystals interfered with the topography of the film, and consequently, in its roughness. This can explain the indirect effect of the CF concentration in the film gloss. Moreover, the micrographs of the cryo-fractured samples, which allow the analysis of the internal microstructure of the films showed that, in overall, the CF were well dispersed and well consolidated in the film matrix, in spite of the presence of some fracture where fibers cross the fracture lines (Fig. 6). There was no
evidence of bright white spots, characteristic of the cellulose fiber when agglomerated (Azizi Samir et al., 2005). Besides, the CF did not cause the occurrence of cracks and pores in the films. These results therefore confirm the results of the mechanical properties. Similar micrograph can be observed in the paper on rice starch based films reinforced with cellulose fibers (Dias et al., 2011).
3.2.8. Fourier transform infrared spectroscopy (FTIR) of films These FTIR analyses demonstrated some changes with the addition of CF when compared to the film without fibers, as shown in Fig. 7. One of the differences is related to the disappearance of the peak at 1191 cm−1 and a significant decrease of the peak at 1096 cm−1 . This peak related to the C O stretching and the presence of glycerol in the sample. Thus, it can be suggested that with the addition of CF, the ratio glycerol/CF decrease and in some manner this behavior affect this band. In addition, the peak at 1042 cm−1 presented small displacements after the CF addition, appearing in the region 1035 cm−1 . This displacement can be explained by interactions between the CF and the matrix (Bergo and Sobral, 2007; Ramos et al., 2013; Saxena et al., 2011). The overall behavior for films with 7.5% of cellulose, where the spectrum was displaced to high when compared with others films, was due to a different attenuation.
Fig. 7. Fourier transformed infrared (FTIR) spectra of the freeze-dried castor bean cake protein based films reinforced with cellulose fibers in different concentrations.
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to the interaction between the fiber and the plasticizer, since the composite is less plasticized than the pure matrix. The concentration of CF did not appreciably affect the melting temperature (Tm ) of the FDCBP based films, which remained around 87 ◦ C, neither it enthalpy, which remained around 2 J/g (Table 2). 4. Conclusion
Fig. 8. Differential scanning calorimetry thermograms of the freeze-dried castor bean cake protein based films with (A) 0%, (B) 2.5%, (C) 5%, (D) 7.5%, (E) 10%, (F) 12.5 g of fiber/100 g protein.
Table 2 Thermal properties of freeze-dried castor bean protein films reinforced with cellulose fibers. Cfibers 0 2.5 5 7.5 10 12.5
Tg (◦ C)(1◦ scan) −50.48 −47.21 −48.23 −48.27 −46.29 −46.33
± ± ± ± ± ±
Tg (◦ C)(2◦ scan) a
2.50 3.00a 2.33a 4.01a 0.90a 2.29a
−52.28 −49.84 −51.44 −51.32 −48.27 −47.94
± ± ± ± ± ±
a
1.49 1.82a 0.62a 4.98a 2.08a 2.22a
Tm (◦ C) 89.56 77.20 87.65 88.57 89.58 89.20
H(J/g) ± ± ± ± ± ±
a
4.10 6.17a,b 2.75a,b 0.89a,b 6.69a 1.36a
2.26 3.22 1.91 1.91 2.10 1.96
± ± ± ± ± ±
a
0.08 1.42a 0.55a 0.07a 0.13a 0.28a
Cfibers : cellulose fiber concentration (g/100 g protein); Tg : Glass transition temperature; Tm : melting temperature; H: melting enthalpy. Different lowercase letters (a and b) in the same column show significant difference for the same parameter.
The band located at 3277 cm−1 corresponded to free water (or amide III), while amide III corresponded to vibrations involving C N and NH in-plane of amide groups attached (Bergo and Sobral, 2007; Khan et al., 2012). The bands at 1635 and 1538 cm−1 corresponded to amide I and amide II, respectively. Amide I mode is a C O stretching, and amide II comes from N H vibrational modes and C N stretching (Bergo and Sobral, 2007). The bands found in the region 2926 and 2868 cm−1 corresponded to the C H symmetric and asymmetric vibrations (Bergo and Sobral, 2007; Chen et al., 2012). 3.2.9. Differential scanning calorimetry (DSC) A glass transition followed by a subtle endothermic peak can be observed in the DSC curves during the first scan (Fig. 8). In the second scan, only glass transition (Tg ) could be observed. These behaviors are usual for partially crystalline material (Sobral et al., 2001). Moreover, the Tg of the films slightly increased depending on the CF concentration (Table 2), both in the first scan as in the second scan. This increase in Tg of the composite films was a consequence of the strong interactions of the fibers with the biopolymer matrix, corroborating with results from microstructure analysis and mechanical properties. These interactions reduced the molecular mobility of the proteins, thereby causing an increase in Tg . In a study on thermoplastic corn starch based films reinforced with eucalyptus pulp fiber, Curvelo et al. (2001) observed an increase in Tg due to the fiber addition (−55 to −45 ◦ C), which was attributed
The load of cellulose fibers improved the mechanical properties of films produced with the freeze-dried castor bean cake protein. The addition of fibers increased the puncture force, tensile strength, and elasticity and decreased the elongation at break of the films. These behaviors were possible because fibers were well dispersed in the protein matrix. Moreover, the cellulose fiber load did not affect others important parameters, such as thickness, humidity, solubility in water, and water vapor permeability. The presence of fibers changed the surface of the material, increasing roughness, evaluated by micrographs, and had influence on film gloss, making it less bright. The fiber addition also affected the total color difference and opacity of the films. Considering the mechanical properties associated the darkness and high opacity of this material, we consider that it can be used in agriculture, as bags for planting seedling when small amount of soil is required due to limitations on mechanical properties. Acknowledgements The authors thanks to São Paulo Research Foundation (FAPESP) for the Grant (08/11341-5), and postdoctoral fellowship of H.N.M.C. (09/10172-8); CAPES for de MS fellowship of T.G.O. and CNPq for the MS fellowship of G.L.A.M. and to Prof. Holmer Savastano Jr. and team, for the samples of fibers. References A.O.A.C, 1995. Official Methods of Analysis. Association of Official Analytical Chemists, Arlington. Agoda-Tandjawa, G., Durand, S., Berot, S., Blassel, C., Gaillard, C., Garnier, C., Doublier, J.L., 2010. Rheological characterization of microfibrillated cellulose suspensions after freezing. Carbohydr. Polym. 80, 677–686. Alves, P.M., Carvalho, R.A., Moraes, I.C.F., Luciano, C.G., Bittante, A.M.Q.B., Sobral, P.J.A., 2011. Development of films based on blends of gelatin and poly(vinyl alcohol) cross linked with glutaraldehyde. Food Hydrocolloids 25, 1751–1757. Arias, A., Heuzey, M.-C., Huneault, M.A., 2012. Thermomechanical and crystallization behavior of polylactide-based flax fiber biocomposites. Cellulose 20, 439–452. Azizi Samir, M.A.S., Alloin, F., Dufresne, A., 2005. Review of recent research into cellulosic whiskers, their properties and their application in nanocomposite field. Biomacromolecules 6, 612–626. Bergo, P., Sobral, P.J.A., 2007. Effects of plasticizer on physical properties of pigskin gelatin films. Food Hydrocolloids 21, 1285–1289. Bittante, A.M.Q.B., Lacerda, R.S., Oliveira, T.G., Makishi, G.L.A., Costa, P.A., Chambi, H.N.M., Gomide, C.A., Sobral, P.J.A., 2014. Properties of biodegradable films made with proteins extracted from castor bean (Ricinus communis) cake: effect of protein extraction pH. Chem. Eng. Trans. 37, 751–756. Bodirlau, R., Teaca, C.-A., Spiridon, I., 2013. Influence of natural fillers on the properties of starch-based biocomposite films. Composites Part B 44, 575–583. Carvalho, R.A., Grosso, C.R.F., 2004. Characterization of gelatin based films modified with transglutaminase: glyoxal and formaldehyde. Food Hydrocolloids 18, 717–726. Chambi, H.N.M., Lacerda, R.S., Makishi, G.L.A., Bittante, A.M.Q.B., Gomide, C.A., Sobral, P.J.A., 2014. Protein extracted from castor bean (Ricinus communis L.) cake in high pH results in films with improved physical properties. Ind. Crops Prod. 61, 217–224. Chen, X., Yu, J., Zhang, Z., Lu, C., 2011. Study on structure and thermal stability properties of cellulose fibers from rice straw. Carbohydr. Polym. 85, 245–250. Chen, D., Lawton, D., Thompson, M.R., Liu, Q., 2012. Biocomposites reinforced with cellulose nanocrystals derived from potato peel waste. Carbohydr. Polym. 90, 709–716. Curvelo, A.A.S., Carvalho, A.J.F., Agnelli, J.A.M., 2001. Thermoplastic starch-cellulosic fibers composites: preliminary results. Carbohydr. Polym. 45, 183–188. Dias, A.B., Müller, C.M.O., Larotonda, F.D.S., Laurindo, J.B., 2011. Mechanical and barrier properties of composite films based on rice flour and cellulose fibers. LWT – Food Sci. Technol. 44, 535–542.
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Cellulose fiber reinforced biodegradable films based on proteins extracted from castor bean (Ricinus communis L.) cake T.G. Oliveira, G.L.A. Makishi, H.N.M. Chambi, A.M.Q.B. Bittante, R.V. Lourenc¸o, P.J.A Sobral ∗ Department of Food Engineering – FZEA/USP, Av. Duque de Caxias Norte, 225, 13635-900 Pirassununga, SP, Brazil
a r t i c l e
i n f o
Article history: Received 17 September 2014 Received in revised form 17 January 2015 Accepted 19 January 2015 Keywords: Biopolymers Composite Thin material Mechanical properties Microstructure
a b s t r a c t The aim of this study was to develop films based on proteins extracted from castor bean (Ricinus communis L.) cake, reinforced with cellulose fibers for use in agriculture, as bags for planting seedlings. The specific aims was to study the effect of fibers concentration on the mechanical properties, color, opacity, gloss, moisture, solubility in water, water vapor permeability (WVP), microstructure, thermal properties, and chemical structure through Fourier transform infrared spectroscopy (FTIR). Proteins were extracted from castor bean cake in a reactor and then freeze-dried. The cellulose fibers were dispersed in water using a high-speed stirrer. The films were produced by dehydration of film-forming solutions (FFS) prepared with the freeze-dried protein (6 g/100 g FFS), cross linker (5 g glyoxal/100 g protein), plasticizer (30 g glycerol/100 g protein), and fibers (0; 2.5; 5; 7.5; 10; and 12.5 g cellulose fibers/100 g protein). The fiber addition had no effect on thickness, humidity, solubility in water and water vapor permeability of the films. In contrast, an increase in puncture force, tensile strength and elastic modulus, and a decrease in the elongation at break were observed as a function of fiber concentration. The fiber addition also affected color, opacity and gloss of the films. Scanning electron microscopy analysis showed that the cellulose fibers were well dispersed in the film matrix, explaining its effect on the mechanical properties of the films. The analysis by Fourier transform infrared spectroscopy (FTIR) corroborated these results. The main conclusion of this study is that the load of cellulose fibers improved the mechanical properties of films produced with the freeze-dried castor bean cake protein. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Environmental concerns associated with plastic waste management have emphasized the importance of developing biodegradable materials to reduce the problems of disposal of plastic waste (Fang et al., 2005). A potential material for the production of biodegradable plastics is the films based on biopolymers such as proteins and polysaccharides, which besides their biodegradable nature, are from renewable resources (Sobral, 2000). Fibers are used as reinforcement materials in biodegradable films to reduce costs and improve it mechanical properties (Kunanopparat et al., 2008; Salgado et al., 2008). Several studies on composites reinforced with vegetable fibers can be found in literature, including wheat gluten based films reinforced with hemp (Kunanopparat et al., 2008), wheat flour based films reinforced with cotton fiber (Dobircau et al., 2009), wheat gluten and fiber
∗ Corresponding author. Tel.: +55 19 35654192; fax: +55 19 35654114. E-mail address: [email protected] (P.J.A Sobral). http://dx.doi.org/10.1016/j.indcrop.2015.01.036 0926-6690/© 2015 Elsevier B.V. All rights reserved.
˜ wheat straw based films (Montano-Leyva et al., 2013), corn starch based films reinforced with keratin, lignin and firs cellulose fibers (Bodirlau et al., 2013), composites of soluble potato starch or corn starch reinforced with sugarcane fibers (Gilfillan et al., 2012), cassava starch based films reinforced with wheat straw fibers (Famá et al., 2009), flax-fiber reinforced PLA biocomposites films (Arias et al., 2012), among others. Among the vegetable fibers, the cellulose fiber is most privileged in studies on biopolymer based films. A highly purified wood pulp composed of 92–98% cellulose, known as dissolved pulp was used in the manufacture of cellulose-derived products (Wertz et al., 2010), and is the most common raw material of microfibrils cellulose (MFC) that can be isolated by employing well-known mechanical treatment methods such as homogenization, microfluidization, microgrinding, and cryocrushing. The stability of its structure confers low solubility of cellulose in almost all reagents and great mechanical strength to the microfibrils (Agoda-Tandjawa et al., 2010; Rezayati Charani et al., 2013; Silva et al., 2008). The cellulose fiber has been applied to produce composites of polyurethane resin derived from castor bean oil (Miléo et al., 2011), starch based
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films (Müller et al., 2009), composite films from galactoglucomannans (Mikkonen et al., 2011) and rice flour based films (Dias et al., 2011). The castor bean (Ricinus communis L.) is an oilseed with high economic value because it presents a well-defined market for the oil. Currently, great interest has been given to castor bean cake generated during oil extraction process for its high protein content, which can reach 40% (Lacerda et al., 2014; Silva et al., 2012; Visser et al., 2011). The castor bean cake may be a viable alternative for the development of biodegradable films, contributing to the success of this biodiesel agribusiness (Makishi et al., 2013; Bittante et al., 2014). But considering that this protein was obtained by solubilization, the major peptide fractions was around 40 and 20 kDa (Lacerda et al., 2014). Then, it could be interesting some degree of modification for increase it molecular weight to guarantee a good film-forming property. Chambi et al. (2014) modified that proteins using tannic acid and produced very workable films. And Makishi et al. (2013) produced films of similar proteins cross linked with glutaraldehyde and glyoxal and observed that glyoxal implied in films with improved mechanical properties and lower film solubility than glutaraldehyde. These behaviors can be explained by the high content of arginine in the protein, which react with glyoxal (Makishi et al., 2013). These materials could have applications in agriculture, particularly as bags for planting seedlings (Bittante et al., 2014). Conventionally, these bags are produced using low-density polyethylene (LDPE) films, which have good physical properties, principally it high mechanical resistance (tensile strength = 34 MPa) (Chambi et al., 2014). However, it is well known that the LDPE is not biodegradable and its bags are difficult to recycle due to the large amount of organic matter adhered to material. The aim of this paper was to develop films based on proteins extracted from the castor bean (R. communis L.) cake reinforced with cellulose fibers interesting for using as bags for plants seedling. Specifically, it was studied the effect of fiber concentration on the mechanical properties (puncture and tensile tests), color, opacity, gloss, humidity, solubility in water, water vapor permeability, microstructure, thermal properties, and chemical structure through Fourier transform infrared spectroscopy. 2. Material and methods Castor bean cake (CBC) was supplied by Azevedo Óleos Indústria e Comercio de Óleos Ltda (Itupeva-SP). Sodium hydroxide and glycerol were purchase from Synth and glyoxal from Sigma. Cellulose fibers (CF) are conventional bleached eucalyptus kraft pulp from Votorantim Cellulose and Paper, Brazil provided from Construction and Environment Group, Faculty of Animal Science and Food Engineering (Savastano et al., 2009). Similar fibers were used to produce starch-based composite films (Dias et al., 2011). 2.1. Proteins extraction from castor bean cake Extractions of the protein from CBC were performed in a 4.5 L reactor (Bio-Tec-V, TECNAL) with controlled temperature (50 ◦ C), pH (12) and agitation (400 rpm) (Lacerda et al., 2014; Makishi et al., 2013). The freeze-dried extracts were analyzed for protein determination by the classical Kjeldahl method (A.O.A.C, 1995). More data on freeze-drying conditions and on the composition of the extracted freeze-dried proteins can be found in a paper published previously (Makishi et al., 2014). 2.2. Cellulose fibers dispersion Cellulosic pulp was dispersed in water by mechanical stirring (3000 rpm for 5 min) (Mármol et al., 2013). The excess water was
removed by manual clamping filtration producing the CF. The humidity of this dispersed pulp was determined by oven drying at 105 ◦ C for 24 h. 2.3. Films preparation The films were produced by casting process at room temperature (22 ± 1 ◦ C). Film-forming solutions (FFS) were prepared with five different CF concentrations (0; 2.5; 5; 7.5; 10 and 12.5 g cellulose fibers/100 g protein). For this preparation, firstly, the CF were dispersed in distilled water under continuous stirring (400 rpm) for 5 min, and then, the freeze-dried Castor bean protein (FDCBP) was slowly added into this CF dispersion under continuous stirring for more 30 min. After FDCBP dispersion, the cross linker (5 g glyoxal/100 g of protein) was added. After 5 min, the plasticizer was also added (30 g glycerol/100 g protein) always under constant magnetic stirring, for 1 min. The FFS were applied to Petri dishes (150 × 15 mm) (Bioplass) and dried in an oven with forced air circulation (Marconi, MA037) at 30 ◦ C and controlled relative humidity (55–65%), for 16 h (Sobral et al., 2001). Before characterizations, the films were conditioned in desiccators containing saturated solutions of Mg(NO2 )2 (52.5% relative humidity) at 25 ◦ C for at least 7 days, except for the samples for scanning electron microcopy and atomic force microscopy analyses, which were conditioned in desiccators with NaOH and in silica gel, respectively, for 7 days. 2.4. Characterization of the cellulose fibers CF were characterized by scanning electron microscopy (SEM), (Hitachi, TM3000; Tokyo, Japan), with a tungsten electron source at 10 kV. Aliquots of a CF solution (1 g CF/50 g of water) were deposited directly onto the carbon tape and dried for 24 h in an oven. 2.5. Characterization of the films All characterizations were carried out in triplicate, at least. 2.5.1. Films thickness The film thickness was determined using a digital micrometer (0.001 mm; Mitutoyo) with a 6.4 mm diameter probe, averaging ten different positions (Sobral et al., 2001). 2.5.2. Humidity and solubility in water The humidity of the films, expressed as g of water/100 g of wet material, was determined by oven drying at 105 ◦ C for 24 h (Gontard et al., 1994). And the solubility in water of the films was determined at 25 ◦ C by immersing the films (d ∼ 2 cm) in distilled water (50 ml) under slight stirring with the aid of a shaker (Marconi, MA141) for 24 h, according to Gontard et al. (1994). The material was filtered through filter paper (Nalgon), weighed and then dried in an oven at 100 ◦ C for 24 h for determination of dry weight. Considering that the initial dry mass of the samples was known, the solubility of the films was calculated as dissolved dry mass. 2.5.3. Mechanical properties The mechanical properties of the films were determined by tensile (tensile strength, elongation at break and elastic modulus) and puncture (puncture force and deformation) tests, using a texturometer TA.XT2i (TA instruments, Surrey, UK), at room temperature (22–25 ◦ C). For the tensile tests, film samples were cut into rectangles (15 mm × 100 mm) and fixed on the grips, separated by 50 mm. These samples were submitted to traction at 1 mm/s (Thomazine et al., 2005). For puncture tests, the films were cut into 46 mm-diameter circles, fixed on a cell, and submitted to perforation by a cylindrical
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probe (d = 3 mm), moving at 1 mm/s (Sobral et al., 2001). The force and displacement at puncture were measured. Puncture deformation (PD, %) was calculated from the distance traveled by the probe to the puncture point, using Eq. (1).
PD(%) = [
(d2 + l02 ) − l0 l0
] × 100
(1)
where l0 is the cell radius (23 mm) and PD: Puncture deformation (%). 2.5.4. Water vapor permeability The water vapor permeability (WVP) was determined gravimetrically, at 25 ◦ C, according to Thomazine et al. (2005). The samples were fixed on aluminum cell containing silica gel (0% RH) and placed in a desiccator containing distilled water (100% RH). These cells were weighed (±0.01 g) daily for 7 days to guarantee the steady state permeation, using a semi-analytical balance (Marte, AS2000, Sao Paulo, Brazil). 2.5.5. Color and opacity The color of the films was determined with a colorimeter Miniscan XE (HunterLab) working with D65 (daylight) lamp, at 10◦ , and measuring with an opening of 30 mm. For color determination, the films were placed on the surface of the standard white plate and de parameters L*, a*, and b* were measured (Gennadios et al., 1996; Kunte et al., 1997). The total color difference (E*) was calculated using Eq. (2): E ∗ =
L∗2 + a∗2 + b∗2
(2)
where L* = L*sample – L*standard (92.58 ± 0.92); a* = a* sample – a* standard (−0.88 ± 0.06); b* = b* sample – b* standard (1.26 ± 0.02). The opacity was measured using the same equipment and computer program used in color measurements. Film opacity was determined according to the Hunterlab method, in the reflectance mode (Sobral, 2000). Opacity (Y) was calculated from the relationship (Y = Yb/Yw) between the opacity of the film superimposed on the black standard (Yb), and that of the film superimposed on the white standard (Yw). 2.5.6. Gloss The gloss of the film surfaces was determined using the Rhopoint NGL 20/60 glossmeter, at angle of 20◦ (Alves et al., 2011; Moraes et al., 2008). The measurements were performed on both surfaces of the films.
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(Carvalho and Grosso, 2004). Both drying surfaces and that in contact with the support were analyzed. The used SEM was coupled to an EDS (energy dispersive spectroscopy detector).
2.5.8. Atomic force microscopy (AFM) These analyses were carried out using an atomic force microscope (model NT-MDT Solver Next brand), equipped with a software for images analysis (New Model 3.1.0 program PX). The films were cut into areas of 2 × 2 cm for use in the AFM. The equipment was operated in tapping mode using NSG tip. The images were recorded using scanning rates of 0.5 Hz. The nominal spring constant and nominal resonant frequency was 11.8 N/m and 240 kHz, respectively.
2.5.9. Fourier transform infrared spectroscopy (FTIR) Fourier transform infrared spectroscopy (FTIR) spectra were recorded using a PerkinElmer spectrometer Spectrum One (PerkinElmer one) equipped with accessory UATR, according to Vicentini et al. (2005). The spectra were recorded in the range of 4000–400 cm−1 with a resolution of 2 cm−1 . Data were analyzed using the Spectrum One (Spectrum One version 5.3) software.
2.5.10. Thermal properties The films thermal properties determination was accomplished by differential scanning calorimetry using a DSC TA 2010 controlled by a TA 5000 module (TA instruments, USA) with quench cooling accessory (Sobral et al., 2001). Aliquots were weighed (∼10 mg) in a precision balance (±0.0001 g) (Scientech, SA210) and placed in hermetic aluminum pans. The system was heated at a rate of 5 ◦ C/min, between −150 and 150 ◦ C in an inert atmosphere (45 ml/min N2 ). The reference was a empty pan. The glass transition temperature (Tg ) was calculated as the inflexion point of the baseline, caused by the discontinuity of the specific heat of the sample. The melting temperature (Tm ) was calculated where the endothermic peak occurred. The enthalpy (Hm) of the sol–gel transition was determined from the endothermic peak area. The properties were calculated with the help of V1.7F Universal Analysis (TA Instruments) software (Sobral et al., 2001).
1.2. Statistical analysis 2.5.7. Scanning electron microscopy The microstructure of films were analyzed using a scanning electron microscope (SEM), (Hitachi TM3000; Tokyo, Japan) with a tungsten electron source at 5 kV, without previous treatment
The results were analyzed using ANOVA and Tukey’s mean comparison test (p ≤ 0.05) was performed using the statistical program “Statistical Analysis Systems” – SAS.
Fig. 1. Scanning electron micrographs of cellulose fibers: (A) cellulose pulp fiber [800× magnification] and, (B) dispersed cellulose fiber [1200× magnification].
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Fig. 2. Effect of cellulose fiber concentration (%) on total difference of color (E*) of freeze-dried castor bean protein based films (– calculated by the Eq. (3)), and on gloss at 20◦ ( side in contact with the plate, side in contact with the air; – calculated by the Eq. (4) and, – and by the Eq. (5). Different letters show significant difference for the same parameters.
3. Results and discussion
composites with pulp cellulose fibers (∼15%) by Curvelo et al. (2001). The solubility in water of the FDCBP based films reinforced with varied from 35.9 to 41.4%, with no significant difference (p > 0.05) among treatments, having an average of 39.33 ± 1.89%. Both the control films and those containing CF maintained their integrity even after 24 h of water immersion, without separation between the matrix and fibers, showing that the intermolecular networks remained unchanged and only low molecular peptides and plasticizer could have solubilized (Wittaya, 2009). This behavior is important for the biodegradation process in soil. Bags based on LPDE are not soluble in water.
3.1. Characterization of fibers The CF presented oval shape and small pores on its surface, formed by grouped fibrils (Fig. 1). The diameters of the CF ranged from 8 to 12 m. Similars results can be found in the work of Chen et al. (2011) and Lu et al. (2014). Savastano et al. (2009) worked with similar cellulose fibers from Eucaliptus grandi and observed fiber length of 0.66 mm and an aspect ratio of 61. They also found a kappa number, what is an indication of residual lignin content in chemical pulps, of 6.1 that indicates the presence of a small content of lignin in the surface of cellulose fibers. According to Dias et al. (2011), the hydroxyl groups of lignin can interact with glycerol, acting as an interfacial compatibilizer between fibers and the matrix, leading to films with higher tensile strength.
3.2.3. Water vapor permeability (WVP) The CF addition in the FDCBP matrix did not statistically affect (p > 0.05) the WVP of films, which remained around 0.73 ± 0.06 g mm/h m2 kPa. Quite possibly, the presence of pores, as discussed in the microscopy results, may have contributed to this behavior. Similar behavior was observed in cassava starch based films reinforced with cellulose fibers, but this behavior was explained by the small variation in water solubility inside films and diffusion coefficient of the films (Müller et al., 2009). Unfortunately, the load with cellulose was not able to transform the material in a high water vapor barrier material, as the LPDE. Nevertheless, this is not necessarily a limitation property for application as bags for plating seedling.
3.2. Characterization of films 3.2.1. Visual aspects and thickness The films were easily removed from the Petri dishes, and had good flexibility and were ease of cutting regardless of the concentrations of CF. Films without CF were brighter than the films containing CF, once they had a smoother texture than the other films. Among the films with CF, visual differences were not observed as a function of the fiber concentration. The presence of CF had no significant (p > 0.05) effect in the film thickness, which remained around a mean value of 0.127 ± 0.004 mm. The films thickness was higher than films based on LPDE for bags for plant sendling, which usually remain around 0.060 mm (Chambi et al., 2014).
3.2.4. Color and opacity The color parameters were measured only on the drying surface of the films, i.e., on the upper surface. The mean L*, a* and b* values were 15.47 ± 1.63; 26.29 ± 1.47; and 19.35 ± 1.24, respectively, with no significant difference (p > 0.05) among treatments. Such values were considered high for films, and imply dark materials that could be interesting to use in agricultural domain. On another hand, the total color difference (E*), which expresses the color of the material, decreased linearly (Eq. (3)) as a function of the CF concentration (Fig. 2). The color parameters of the FDCBP used as raw material were high (L* = 52.42 ± 0.01,
3.2.2. Humidity and solubility in water The addition of CF did not affect (p > 0.05) the films humidity, which had an average value of 14.35 ± 0.57%, allowing to suggest that CF did not cause changes in the films hygroscopicity. The humidity values were close to those found in corn starch based
Table 1 Elastic modulus, puncture deformation and opacity of freeze-dried castor bean protein films reinforced with cellulose fibers as a function of cellulose concentration (Cfibers ). Cfibers (g/100 g protein) 0 Elastic modulus(MPa/%) Puncture deformation(%) Opacity
2.5
1.43 ± 0.18 7.50 ± 0.26a 15.91 ± 1.02b
b,c
5
1.32 ± 0.38 6.87 ± 0.45a,b 28.56 ± 12.69a,b c
7.5
1.80 ± 0.32 7.24 ± 0.54b,c 30.91 ± 7.9a,b
Different lowercase letters in the same line show significant difference for the same parameter.
a,b
10
1.82 ± 0.29 6.65 ± 0.33c 29.94 ± 9.67a,b a,b
12.5
2.05 ± 0.48 6.66 ± 0.26c 33.65 ± 7.49a,b a
1.70 ± 0.40a,b,c 6.58 ± 0.36c 45.31 ± 12.33a
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Fig. 3. Effect of cellulose fiber concentration (%) on tensile strength and elongation at break of freeze-dried castor bean protein based films (– calculated by the Eq. (6) and, – by the Eq. (7)), and on puncture force of freeze-dried castor bean protein based films (– calculated by the Eq. (8). Different letters show significant difference for the same parameter.
a* = 6.05 ± 0.04, b* = 18.44 ± 0.03) because it is a dark material, due the presence of pigments as quinone (Lacerda et al., 2014), in contrast to cellulose fibers, which, being white, presented low values (L* = 94.02 ± 2.05, a* = 0.05 ± 0.01, b* = 4.27 ± 0.05). Thus, the
decrease of the total color difference of the FDCBP based films must have been due to the mixture of both products. E ∗ = −0.35Cfiber + 86.5(R2 = 0.9704)
(3)
Fig. 4. Scanning electron micrographs of drying surface (A1 and B1) and of support contact surface (A2 and B2) of freeze-dried castor bean cake protein based films reinforced with cellulose fibers at (A1 and A2) 0 and (B1 and B2) 12.5 g of fiber/100 g protein. 500× magnification.
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Fig. 5. Micrographs of a salt crystal in freeze-dried castor bean cake protein based films obtained by (A) scanning electron microscopy and (B) atomic force microscopy.
where E*:Total color difference and Cfiber : Fiber concentration (%). The opacity of the films varied (p < 0.05) with the CF concentration, ranging from 15.9 to 45.3 when the CF concentration increased from zero to 12.5% (Table 1). These values can be considered a high value for protein films, which means that these films can be considered almost as opaque ones. Usually, bags for planting seedling are dark and opaque, to avoid the sun penetration in soil. But in despite of the reduction in the total color difference provoked by the cellulose load, this material can be considered as dark (E* > 82), and thus, having potential to be used as bags for plating seedling. The effect of cellulose load was positive and corroborate with this last statement. 3.2.5. Gloss The gloss of the films surfaces was determined at an angle of 20◦ . For all films, a decrease in gloss was observed in both the drying surface and on the surface in contact with the support, which was dependent of CF concentration (Fig. 2). In the drying surface, the gloss values decreased linearly (Eq. (4)) from 9.4 to 0.4 when the CF concentration increased from zero to 12.5%, while for support contact surface this decrease was (Eq. (5)) from 14.7 to 3.4. No data on gloss of bags based on LPDE was found. Drying surface : G = −0.69Cfiber + 8.4(R2 = 0.9547)
(4)
Contact surface : G = −0.86Cfiber + 14.0(R2 = 0.9544)
(5)
where G: Gloss
(20◦ )
and Cfiber : Fiber concentration (%).
3.2.6. Mechanical properties 3.2.6.1. Tensile tests. The addition of CF in the film matrix provoked a linear increase (Eq. (6)) in tensile strength as a function of CF concentration, evidencing a strong positively influence of the CF on the mechanical properties of the composites (Fig. 3). The maximum CF concentration resulted in an increase of approximately 42% in the tensile strength of the composite films as compared to control, suggesting that the FDCBP based composites reinforced with cellulose fiber presented adequate dispersion of fiber in the biopolymer matrix (Miléo et al., 2011). Nevertheless, the improved material (TS = 13 MPa) was always less resistant then the LPDE-based bags. But in spite of that, the FDCBP based composite could be indicated to bags for planting with need low quantity of soil. TS = 0.35Cf iber + 8.4(R2 = 0.865)
(6)
where TS: Tensile strength (MPa) and Cfiber : Fiber concentration (%). Dobircau et al. (2009) observed a similar behavior working on rice flour starch based films, where the increase in tensile strength
was attributed to good dispersion of the fiber in biopolymer matrix, demonstrating that the stress transfers from the fiber to the matrix occurred properly. Dias et al. (2011) studied the effect of the addition of cellulose fibers on rice flour based films, and found that the addition of 0.3 g fibers/g dried flour (plasticized with 0.3 g glycerol/g flour) improved the mechanical properties of the films. Similar results were obtained by Miléo et al. (2011), who studied castor oil composites reinforced with sugarcane straw cellulose fibers at various concentrations (0, 5, 10, 15 and 20% w/w), and also observed an increase in the tensile strength. Regarding the elongation at break, it was noted that this mechanical property decreased exponentially (Eq. (7)) with increasing of CF concentration in FDCBP based films (Fig. 3), suggesting that the strong protein/fibers interactions restricted the movement of protein chains identical to an anti-plasticizing behavior. Increasing the fiber concentration in the films resulted in a decrease in the elongation at break from 110.67 to 23.26%, as compared to the films with 12.5% cellulose fiber in relation to the control. The observed decreasing in FDCBP based films EB do not constitute a limitation for use as bag for planting seedling. EB = 92.5e−0.12Cf iber (R2 = 0.8963)
(7)
where EB: Elongation at break (%) and Cfiber : Fiber concentration (%). The decrease in the elongation at break as a function of increasing CF concentration was also observed in rice flour based films reinforced with cellulose fibers plasticized with 0.3 g glycerol/g flour, with values from 8.8 to 66.4% when 0.3 g fibers/g flour was added (Dias et al., 2011). In cassava starch based films reinforced with cellulose fibers, the elongation at break decreased by up to 18 times in films with 0.5 g fibers/g starch when compared with the pure matrix (Müller et al., 2009). The addition of CF as reinforcement in FDCBP based films resulted in an increase in the elastic modulus of the films (Table 1), without necessarily follow a monotonic behavior. The maximum value for this module was observed for films with 10% in CF, representing an increase of 30.2% as compared to the control film. This behavior is in agreement with studies on polyurethane from castor oil films reinforced with cellulose fiber from sugarcane bagasse (Miléo et al., 2011). 3.2.6.2. Puncture tests. The results of the mechanical properties determined by puncture tests corroborated with the results of tensile strength. The puncture force showed a linear increase (Eq. (8)) as a function of CF concentration (Fig. 3). PF = 0.38Cfiber + 10.7(R2 = 0.9704)
(8)
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Fig. 6. Scanning electron micrographs of cryofractured surfaces of freeze-dried castor bean cake protein based films reinforced with cellulose fibers at (A) 0 and (B) 12.5 g of fiber/100 g protein. 1200× magnification.
where PF: Puncture force (N) and Cfiber : Fiber concentration (%). Similar to the results for tensile tests, the puncture deformation decreased with increasing CF concentration (Table 1), although this decrease was less pronounced than in the tensile test. 3.2.7. Microstructure of the films The scanning electron micrographs of films surfaces (Fig. 4) presented circular microstructures, not visible to the naked eye, and in greater amounts in the drying surface, independent of the CF concentration. These particles were agglomerates of salts, possibly formed from the NaOH used in the protein extraction (Lacerda et al., 2014), that was dragged during drying, thus explaining the highest concentration in the drying surface. And apparently, the amount of these particles in the surface increased with CF concentration in films possible due to the increasing of some porosity in films. Fig. 5 shows these salt crystals more clearly being that AFM microscopy showed that these crystals interfered with the topography of the film, and consequently, in its roughness. This can explain the indirect effect of the CF concentration in the film gloss. Moreover, the micrographs of the cryo-fractured samples, which allow the analysis of the internal microstructure of the films showed that, in overall, the CF were well dispersed and well consolidated in the film matrix, in spite of the presence of some fracture where fibers cross the fracture lines (Fig. 6). There was no
evidence of bright white spots, characteristic of the cellulose fiber when agglomerated (Azizi Samir et al., 2005). Besides, the CF did not cause the occurrence of cracks and pores in the films. These results therefore confirm the results of the mechanical properties. Similar micrograph can be observed in the paper on rice starch based films reinforced with cellulose fibers (Dias et al., 2011).
3.2.8. Fourier transform infrared spectroscopy (FTIR) of films These FTIR analyses demonstrated some changes with the addition of CF when compared to the film without fibers, as shown in Fig. 7. One of the differences is related to the disappearance of the peak at 1191 cm−1 and a significant decrease of the peak at 1096 cm−1 . This peak related to the C O stretching and the presence of glycerol in the sample. Thus, it can be suggested that with the addition of CF, the ratio glycerol/CF decrease and in some manner this behavior affect this band. In addition, the peak at 1042 cm−1 presented small displacements after the CF addition, appearing in the region 1035 cm−1 . This displacement can be explained by interactions between the CF and the matrix (Bergo and Sobral, 2007; Ramos et al., 2013; Saxena et al., 2011). The overall behavior for films with 7.5% of cellulose, where the spectrum was displaced to high when compared with others films, was due to a different attenuation.
Fig. 7. Fourier transformed infrared (FTIR) spectra of the freeze-dried castor bean cake protein based films reinforced with cellulose fibers in different concentrations.
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to the interaction between the fiber and the plasticizer, since the composite is less plasticized than the pure matrix. The concentration of CF did not appreciably affect the melting temperature (Tm ) of the FDCBP based films, which remained around 87 ◦ C, neither it enthalpy, which remained around 2 J/g (Table 2). 4. Conclusion
Fig. 8. Differential scanning calorimetry thermograms of the freeze-dried castor bean cake protein based films with (A) 0%, (B) 2.5%, (C) 5%, (D) 7.5%, (E) 10%, (F) 12.5 g of fiber/100 g protein.
Table 2 Thermal properties of freeze-dried castor bean protein films reinforced with cellulose fibers. Cfibers 0 2.5 5 7.5 10 12.5
Tg (◦ C)(1◦ scan) −50.48 −47.21 −48.23 −48.27 −46.29 −46.33
± ± ± ± ± ±
Tg (◦ C)(2◦ scan) a
2.50 3.00a 2.33a 4.01a 0.90a 2.29a
−52.28 −49.84 −51.44 −51.32 −48.27 −47.94
± ± ± ± ± ±
a
1.49 1.82a 0.62a 4.98a 2.08a 2.22a
Tm (◦ C) 89.56 77.20 87.65 88.57 89.58 89.20
H(J/g) ± ± ± ± ± ±
a
4.10 6.17a,b 2.75a,b 0.89a,b 6.69a 1.36a
2.26 3.22 1.91 1.91 2.10 1.96
± ± ± ± ± ±
a
0.08 1.42a 0.55a 0.07a 0.13a 0.28a
Cfibers : cellulose fiber concentration (g/100 g protein); Tg : Glass transition temperature; Tm : melting temperature; H: melting enthalpy. Different lowercase letters (a and b) in the same column show significant difference for the same parameter.
The band located at 3277 cm−1 corresponded to free water (or amide III), while amide III corresponded to vibrations involving C N and NH in-plane of amide groups attached (Bergo and Sobral, 2007; Khan et al., 2012). The bands at 1635 and 1538 cm−1 corresponded to amide I and amide II, respectively. Amide I mode is a C O stretching, and amide II comes from N H vibrational modes and C N stretching (Bergo and Sobral, 2007). The bands found in the region 2926 and 2868 cm−1 corresponded to the C H symmetric and asymmetric vibrations (Bergo and Sobral, 2007; Chen et al., 2012). 3.2.9. Differential scanning calorimetry (DSC) A glass transition followed by a subtle endothermic peak can be observed in the DSC curves during the first scan (Fig. 8). In the second scan, only glass transition (Tg ) could be observed. These behaviors are usual for partially crystalline material (Sobral et al., 2001). Moreover, the Tg of the films slightly increased depending on the CF concentration (Table 2), both in the first scan as in the second scan. This increase in Tg of the composite films was a consequence of the strong interactions of the fibers with the biopolymer matrix, corroborating with results from microstructure analysis and mechanical properties. These interactions reduced the molecular mobility of the proteins, thereby causing an increase in Tg . In a study on thermoplastic corn starch based films reinforced with eucalyptus pulp fiber, Curvelo et al. (2001) observed an increase in Tg due to the fiber addition (−55 to −45 ◦ C), which was attributed
The load of cellulose fibers improved the mechanical properties of films produced with the freeze-dried castor bean cake protein. The addition of fibers increased the puncture force, tensile strength, and elasticity and decreased the elongation at break of the films. These behaviors were possible because fibers were well dispersed in the protein matrix. Moreover, the cellulose fiber load did not affect others important parameters, such as thickness, humidity, solubility in water, and water vapor permeability. The presence of fibers changed the surface of the material, increasing roughness, evaluated by micrographs, and had influence on film gloss, making it less bright. The fiber addition also affected the total color difference and opacity of the films. Considering the mechanical properties associated the darkness and high opacity of this material, we consider that it can be used in agriculture, as bags for planting seedling when small amount of soil is required due to limitations on mechanical properties. Acknowledgements The authors thanks to São Paulo Research Foundation (FAPESP) for the Grant (08/11341-5), and postdoctoral fellowship of H.N.M.C. (09/10172-8); CAPES for de MS fellowship of T.G.O. and CNPq for the MS fellowship of G.L.A.M. and to Prof. Holmer Savastano Jr. and team, for the samples of fibers. References A.O.A.C, 1995. Official Methods of Analysis. Association of Official Analytical Chemists, Arlington. Agoda-Tandjawa, G., Durand, S., Berot, S., Blassel, C., Gaillard, C., Garnier, C., Doublier, J.L., 2010. Rheological characterization of microfibrillated cellulose suspensions after freezing. Carbohydr. Polym. 80, 677–686. Alves, P.M., Carvalho, R.A., Moraes, I.C.F., Luciano, C.G., Bittante, A.M.Q.B., Sobral, P.J.A., 2011. Development of films based on blends of gelatin and poly(vinyl alcohol) cross linked with glutaraldehyde. Food Hydrocolloids 25, 1751–1757. Arias, A., Heuzey, M.-C., Huneault, M.A., 2012. Thermomechanical and crystallization behavior of polylactide-based flax fiber biocomposites. Cellulose 20, 439–452. Azizi Samir, M.A.S., Alloin, F., Dufresne, A., 2005. Review of recent research into cellulosic whiskers, their properties and their application in nanocomposite field. Biomacromolecules 6, 612–626. Bergo, P., Sobral, P.J.A., 2007. Effects of plasticizer on physical properties of pigskin gelatin films. Food Hydrocolloids 21, 1285–1289. Bittante, A.M.Q.B., Lacerda, R.S., Oliveira, T.G., Makishi, G.L.A., Costa, P.A., Chambi, H.N.M., Gomide, C.A., Sobral, P.J.A., 2014. Properties of biodegradable films made with proteins extracted from castor bean (Ricinus communis) cake: effect of protein extraction pH. Chem. Eng. Trans. 37, 751–756. Bodirlau, R., Teaca, C.-A., Spiridon, I., 2013. Influence of natural fillers on the properties of starch-based biocomposite films. Composites Part B 44, 575–583. Carvalho, R.A., Grosso, C.R.F., 2004. Characterization of gelatin based films modified with transglutaminase: glyoxal and formaldehyde. Food Hydrocolloids 18, 717–726. Chambi, H.N.M., Lacerda, R.S., Makishi, G.L.A., Bittante, A.M.Q.B., Gomide, C.A., Sobral, P.J.A., 2014. Protein extracted from castor bean (Ricinus communis L.) cake in high pH results in films with improved physical properties. Ind. Crops Prod. 61, 217–224. Chen, X., Yu, J., Zhang, Z., Lu, C., 2011. Study on structure and thermal stability properties of cellulose fibers from rice straw. Carbohydr. Polym. 85, 245–250. Chen, D., Lawton, D., Thompson, M.R., Liu, Q., 2012. Biocomposites reinforced with cellulose nanocrystals derived from potato peel waste. Carbohydr. Polym. 90, 709–716. Curvelo, A.A.S., Carvalho, A.J.F., Agnelli, J.A.M., 2001. Thermoplastic starch-cellulosic fibers composites: preliminary results. Carbohydr. Polym. 45, 183–188. Dias, A.B., Müller, C.M.O., Larotonda, F.D.S., Laurindo, J.B., 2011. Mechanical and barrier properties of composite films based on rice flour and cellulose fibers. LWT – Food Sci. Technol. 44, 535–542.
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