Fish gelatin films as affected by cellulose whiskers and sonication

Food Hydrocolloids 41 (2014) 113e118

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Fish gelatin films as affected by cellulose whiskers and sonication Talita M. Santos a, Men de Sá M. Souza Filho b, Carlos Alberto Caceres c, Morsyleide F. Rosa b, João Paulo S. Morais d, Alaídes M.B. Pinto a, Henriette M.C. Azeredo e, * a

Chemical Engineering Department, Federal University of Ceará, Campus Pici, Bloco 709, 60455-760 Fortaleza, CE, Brazil Embrapa Tropical Agroindustry, R Dra Sara Mesquita, 2270, 60511-110 Fortaleza, CE, Brazil c Universidade da Integração Internacional da Lusofonia Afro-Brasileira, Instituto de Engenharias e Desenvolvimento Sustentável, CE 060, Km 51, 62785-000 Acarape, CE, Brazil d Embrapa Cotton, R. Oswaldo Cruz, 1143, Caixa Postal 174, 58428-095 Campina Grande, PB, Brazil e Embrapa e Secretariat for International Affairs, Edifício Embrapa Sede, Prédio CECAT, 3 andar, Parque Estação Biológica, Av. W3 Norte, Brasília, DF CEP 70770-901, Brazil b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 November 2013 Accepted 1 April 2014 Available online 13 April 2014

This study was conducted to evaluate the influence of cellulose whiskers (CW) from cotton linter and sonication on physical properties of glycerol-plasticized tilapia gelatin films produced by casting technique. The tensile strength and elastic modulus of the films have been improved by the addition of CW, while elongation tended to be impaired by CW loading. The barrier to water vapor has also been improved by the whiskers, and the film transparency has been unaffected. The sonication improved modulus, elongation, and transparency of the films, probably by enhancing CW dispersion on the nanocomposite films, and maybe by inducing some conformational changes on the gelatin matrix. FTIR spectroscopy study on films revealed some gelatin conformational changes induced by cellulose whiskers. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Protein Biopolymers Edible films Food packaging Nanotechnology Ultrasound

1. Introduction Biodegradable materials have been increasingly suggested as an alternative to petrochemical polymers for food packaging, which are non-biodegradable and cause several problems related to their accumulation. Biodegradable films are usually made from biopolymers, including polysaccharides and proteins. The main mechanism of formation of protein films involves denaturation of the protein initiated by heat, solvents, or change in pH, followed by association of peptide chains through new intermolecular interactions (Janjarasskul & Krochta, 2010). Gelatin is a substantially pure protein food ingredient, obtained by a mild heat treatment of collagen under acidic or alkaline conditions, when collagen is partially denatured, but recovers part of its triple helix structure upon cooling. On dehydration, films are formed with irreversible conformational changes (Badii & Howell, 2006; Dangaran, Tomasula, & Qi, 2009). The most usual gelatin sources are collagen from animal bones and skin generated as

* Corresponding author. E-mail address: [email protected] (H.M.C. Azeredo). http://dx.doi.org/10.1016/j.foodhyd.2014.04.001 0268-005X/Ó 2014 Elsevier Ltd. All rights reserved.

waste during processing. Although bovine and porcine wastes are the most frequent gelatin sources, there has been an increasing demand for alternative sources, due to the outbreak of bovine spongiform encephalopathy (BSE) as well as religious considerations (Chiou et al., 2009; Eysturskarð, Haug, Elharfaoui, Djabourov, & Draget, 2009; Rahman, Al-Saidi, & Guizani, 2008). Gelatin from fish wastes has been looked upon as a possible alternative to mammalian gelatin. However, cold water fish gelatins have not generated much commercial interest because of their suboptimal physical properties, such as lower storage modulus, as well as lower gelling and melting temperatures, when compared to mammalian gelatin, which is attributed to their lower contents of proline and hydroxyproline (Chiou et al., 2009; Haug, Draget, & Smidsrød, 2004). Warm water fish species, on the other hand, have higher proline and hydroxyproline contents and more similar characteristics to porcine gelatin when compared to cold water species (Karim & Bhat, 2009). Gelatin from several warm fish species have been studied, including tilapia (Oreochromis niloticus) (Jamilah & Harvinder, 2002; Jamilah, Tan, Umi Hartina, & Azizah, 2011; Tongnuanchan, Benjakul, & Prodpran, 2013). The entire substitution of petroleum-derived polymers with biopolymers is not still possible because of the poor overall performance of the latter, suggesting that research is still necessary to

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improve physical properties of biopolymers in order to make such substitution increasingly feasible. One of the most promising ways to improve biopolymer properties is by adding reinforcing nanostructures such as nanocellulose, which has attracted special interest over the past few decades as a biobased reinforcing nanofiller (Dufresne, 2013). Two types of nanoreinforcements may be obtained from cellulose: cellulose microfibrils, and cellulose nanocrystals (also known as cellulose nanowhiskers or whiskers). The microfibrils consist of elongated bundles of cellulose chains which are stabilized through hydrogen bonding, typically with 2e20 nm in diameter and micrometer-sized lengths, and, containing both amorphous and crystalline regions (Azizi Samir, Alloin, & Dufresne, 2005). Cellulose whiskers are the crystalline regions isolated from microfibrils (Lima & Borsali, 2004), and have demonstrated to be an effective nanoreinforcement for biopolymer films (Bras, Viet, Bruzzese, & Dufresne, 2011; Pasquini, Teixeira, Curvelo, Belgacem, & Dufresne, 2010; Siqueira, Abdillahi, Bras, & Dufresne, 2010), drastically enhancing the mechanical properties of biomaterials, especially above the glass transition temperature of the matrix, thanks to the formation of a continuous and rigid network of whiskers, assumed to be governed by a percolation mechanism (Bras et al., 2011). Moreover, cellulose whiskers have been demonstrated to reduce film permeability to water vapor (Follain et al., 2013; Saxena, Elder, & Ragauskas, 2011; Saxena & Ragauskas, 2009). Acoustic cavitation is the mechanism of many of the sonochemical reactions in liquids. In an acoustic field, cavitation bubbles grow and collapse, generating high temperatures within the bubbles and the emission of light (sonoluminescence), and generation large shear forces which can break inter and intramolecular bonds, which in turn may lead to fragmentation of clusters and aggregates (Chandrapala, Zisu, Palmer, Kentish, & Ashokkumar, 2011). Ultrasound technology has been used to improve dispersion of cellulose nanostructures in polymer matrices (Agoda-Tandjawa et al., 2010; Elmabrouk, Wim, Dufresne, & Boufi, 2009). Sonication processes can also affect the structures of some matrices. High-intensity ultrasound using low frequencies (usually 20e100 kHz) can produce high power levels, generating intense pressure, shear and temperature gradients within a material, which can physically disrupt its structure, breaking intermolecular bonds (Leadley & Williams, 2006). Although many studies have been conducted on gelatin films, no previous study has evaluated the effects of ultrasound on fish gelatin films reinforced with different concentration of cellulose whiskers. The objective of this study was to evaluate the influence of the concentration of cellulose whiskers from cotton linter and sonication on physical properties of fish gelatin films. 2. Materials and methods 2.1. Preparation of gelatin and cellulose whiskers A gelatin powder (pH, 5.45; bloom strength, 139 g; protein dry content, 72.15%; ash content, 17.57%) was obtained as described by Souza Filho et al. (2012). Despite the high ash content, the material was not demineralized, since the minerals could have a beneficial effect on the functional properties of gelatin, especially on gelation (Benjakul, Oungbho, Visessanguan, Thiansilakul, & Roytrakul, 2009). The tilapia residue (skins, bones and scales) was washed, immersed in NaCl 0.5% for 15 min, washed again, immersed in 0.2 N acetic acid for 45 min, neutralized in 1 M NaOH, and ground. The ground residue was then submitted to an alkaline treatment (0.2 N NaOH solution on a 1:3 ground residue: NaOH solution under stirring for 45 min), neutralized with 1 M H2SO4, and centrifuged (Hitachi CR 22GIII) for 5 min at 2795  g. The precipitate was

weighted and submitted to an acid treatment (0.2 N H2SO4 solution on a 1:3 precipitate: H2SO4 solution under stirring for 45 min), neutralized with NaOH, and centrifuged as described before. The precipitate was then extracted with water at 45  C (precipitate: water ratio, 1:5) for 2 h, vacuum filtered through 28 mm filter paper, freeze dried, and ground by using an analytical mill (A11 Basic, Ika, Staufen, Germany). The cellulose whiskers (CW) were obtained from cotton linter by acid hydrolysis (Cranston & Gray, 2006; Morais et al., 2013) with aqueous sulfuric acid (60%, w/w) at a 1:20 (w/v) linter/sulfuric acid solution ratio at 45  C, for 1 h, under stirring. The suspension was then centrifuged (Hitachi CR 22GIII) for 15 min at 18,894.2  g, and the precipitate was re-suspended in distilled water and dialyzed with water until a pH of 6e7. The process from centrifugation through dialysis was repeated three times. A final suspension was obtained with a solid content of 3.12% (w/w). The nanocrystals had 12 nm in diameter and 177 nm in length, as measured by transmission electron microscopy (TEM) as described by Morais et al. (2013). 2.2. Film formation Fish gelatin and glycerol were hydrated in distilled water at room temperature (24  C). The quantities were determined so as to obtain the following final concentrations (taking in account the CW suspension, which was added afterward): gelatin, 9.6 g/100 g; glycerol, 25 wt% on a dry gelatin basis. The gelatin aqueous suspension was heated to 50  C under stirring, and maintained at this temperature for 15 min. The gelatin solution was then homogenized by using Ultra Turrax T-25 (Ika, Staufen, Germany) at 10,000 rpm for 10 min, the CW suspension being gradually added to the gelatin solution during the first 2 min. Four film-forming solutions were obtained, with the following CW concentrations (on a dry gelatin basis, w/w): 0 wt%, 5 wt%, 10 wt%, and 15 wt% (CW15). Each film-forming solution was then divided into two equal parts, one of them being submitted to sonication in a 400 W ultrasonic processor (UP400S, Hielscher, Teltow, Germany, with a titanium H22 sonotrode) working at a 24 kHz for 10 min. In the end, there were eight treatments: CW0 (0 wt% CW, no sonication); CW5 (5 wt% CW, no sonication); CW10 (10 wt% CW, no sonication); CW15 (15 wt% CW, no sonication); CW0S (0 wt% CW, sonication); CW5S (5 wt% CW, sonication); CW10S (10 wt% CW, sonication); CW15S (15 wt% CW, sonication), consisting in each film-forming solution submitted or not to sonication. The film-forming solutions were vacuum degassed by using a vacuum pump V-700 (Büchi Labortechnik AG, Flawil, Switzerland) at 30 mbar for 1 h, cast on glass plates, leveled with a draw-down bar to a thickness of 1.0 mm, and placed on a lab bench (24  C, 24 h) to dry. After cooling, dried samples were cut and detached from the surface. Before film characterization, the detached, free-standing samples were conditioned for 40 h at 25  C in desiccators containing a saturated solution of magnesium nitrate in order to maintain the a constant relative humidity of 55%. The water vapor permeability (WVP) determination, with eight replicates, was based on the method E96-00 (ASTM., 2002) at 25  C and 85% RH, using silica gel as the desiccant material. Eight measurements were made within a 24-h period. Film opacity determination (in triplicate) was based on the method described by Irissin-Mangata, Bauduin, Boutevin, and Gontard (2001). Films were cut into rectangular (1  4 cm) shaped strips and placed onto the internal side of a Varian Cary 50 UV VIS spectrophotometer test cell (Agilent Technologies, Santa Clara, CA, USA), perpendicularly to the light beam. The absorbance spectrum (400e800 nm) of film samples were recorded and then film opacity

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was defined as the area under the recorded curve (calculated by the linear trapezoidal rule) and expressed as absorbance units  nanometers (A nm). Tensile properties were measured on film specimens (five replicates), according to D882-00 (ASTM., 2000), in an Emic DL-3000 Universal Testing Machine with a load cell of 100 N, initial grip separation of 0.05 m, and crosshead speed of 10 mm/min (1.67  10 4 m/s). The scanning electron microscopy (SEM) images of the films were taken using a Hitachi TM 3000 scanning electron microscope (Tokyo, Japan), with the samples mounted on an aluminum stub with a double side adhesive. The samples were examined using an accelerating voltage of 5 kV, and a magnification of 400 times. Fourier-transform infrared spectroscopy (FTIR) spectra were recorded to study the effects of the cellulose whiskers on gelatin films in term of molecular and supermolecular structure and organization, in a Varian 660-IR spectrophotometer equipped with an attenuated total reflectance (ATR) sampling accessory, in the wavenumber range from 4000 to 650 cm 1 with a resolution of 4 cm 1 over 25 scans. 3. Results and discussion Fig. 1 presents the effects of CW concentration and sonication on properties of fish gelatin films. The tensile strength of the films was significantly increased by the addition of 5 wt% of CW, but was not improved by further CW adding. Indeed, one of the advantages of nanocomposites over conventional composites is that relatively small filler loadings (typically under 5 wt%) are required as result of the nanometric scale dispersion of the filler in the matrix (Alexandre & Dubois, 2000; Lepoittevin et al., 2002), and that further loading may result in the leveling off or even a decrease of some properties (Lepoittevin et al., 2002; Paul et al., 2003). A similar behavior was observed for elastic modulus of the nonsonicated films, while the modulus of sonicated films was further increased by the addition of CW up to 15 wt%. For sonicated films,

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the elongation tends to be impaired by higher CW concentrations. Overall, gelatin nanocomposites seems to combine high stiffness and good ductility up to a CW loading of 5 wt%; above this loading, the mechanical properties tend to reach a plateau or even to decrease. The sonication improved both modulus and elongation. However, sonication did not significantly affected tensile strength, although previous studies have reported increased tensile strength in nanocomposite films by ultrasonic treatment (Dean & Yu, 2005; He et al., 2006; Zeng, Gao, Wu, Fan, & Li, 2010). The positive effects of sonication on overall tensile properties can be in part ascribed to an enhanced CW dispersion (Boufi, Kaddami, & Dufresne, 2013; Mabrouk, Vilar, Magnin, Belgacem, & Boufi, 2011), increasing the surface area exposed to the polymer (Arora & Padua, 2010; McAdam, Hudson, Liggat, & Pethrick, 2008). Moreover, since sonication has even improved the tensile properties of the unloaded gelatin films, it should have some effect on gelatin itself. According to Liu, Tellez-Garay, and Castell-Perez (2004), the conformational changes on peanut protein can increase the formation of disulfide cross-linking and hydrophobic interactions, thus favoring the tensile properties of peanut protein films. Although those kind of intermolecular interactions are not relevant for gelatin, it is possible that sonication induces some conformational changes in gelatin which result in enhanced tensile properties. While the mechanical properties tend to a plateau above 5 wt% of CW, the water vapor permeability (WVP) was further reduced by increasing CW loadings. Paralikar, Simonsen, and Lombardi (2008) reported that 10 wt% of CW had a better effect on barrier of poly(vinyl alcohol) films to water vapor than 20 wt% of CW, and attributed this to agglomeration of nanocrystals at 20 wt%, which may have provided for channels that allowed increased permeation. Other studies reported decreasing WVP from increasing nanocellulose loadings on protein films (George & Siddaramaiah, 2012; Pereda, Amica, Rácz, & Marcovich, 2011), but those authors studied only low nanocellulose loadings (up to 3e4 wt%). The CW effect on reducing water transmission through films can be attributed to the absence of amorphous areas through which the

Fig. 1. Effect of CW concentration and sonication on properties of fish gelatin films. WVP: water vapor permeability.

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water transmission preferentially occurs (George & Siddaramaiah, 2012; Saxena & Ragauskas, 2009), as well as to the dense composite structure formed by the hydrogen-bonded percolating network of cellulose whiskers (Dufresne, 2013; Follain et al., 2013; Saxena & Ragauskas, 2009). The sonication presented inconsistent effects on WVP, increasing it on films without CW, and decreasing it on films with higher CW loadings. The increase of WVP on unloaded films may have resulted from conformational changes on gelatin. Ultrasound can promote breakdown of hydrogen bonds and Van der Waals interactions in polypeptide chains, changing secondary and tertiary structure of proteins (Barteri, Fioroni, & Gaudiano, 1996; Tian, Wan, Wang, & Kang, 2004), and those changes may be related to increased water solubility and, consequently, water vapor permeability gelatin films. On the other hand, for the films with CW, this effect seemed to be compensated by the sonication effects on improving CW dispersion within the matrix. For comparison with typical petrochemically derived polymer films, the gelatin films obtained in this study presented improved overall tensile properties (namely, higher strength and modulus, but lower elongation) than those reported by Hotta and Paul (2004) for linear low density polyethylene (LLDPE). On the other hand, even the nanocomposite films presented much higher WVP values when compared to those reported for polypropylene by Tihminlioglu, Atik, and Özen (2010). CW did not significantly affect film transparency, corroborating reports from Fortunati et al. (2012) on poly(lactic acid) films added with cellulose nanocrystals. On the other hand, other previous studies reported that cellulose whiskers reduced the transparency of chitosan (Li, Zhou, & Zhang, 2009) and sodium caseinate (Pereda et al., 2011) films. Sonication reduced the film opacity, since it enhanced the dispersion of whiskers (Boufi et al., 2013; Mabrouk et al., 2011), making them less able to scatter light (Petersson, Mathew, & Oksman, 2009). Indeed, the SEM images (Fig. 2) of the films added with 15 wt% cellulose whiskers indicate that the sonication (Fig. 2B) improved the dispersion of the whiskers (decreasing the thickness of whiskers bundles), although the sonicated film still presented some whisker aggregation. This enhanced whiskers dispersion is probably responsible for the improved overall tensile and water vapor barrier properties of sonicated nanocomposite films. Sonication has been also reported to improve the dispersion of other nanoparticles in films, such as nanoclays (Bae et al., 2009; McAdam et al., 2008; Pandey, Bhattacharyya, Gutch, Chauhan, & Pant, 2010), silica (Haldorai, Long, Noh, Lyoo, & Shim, 2011), and nanosilver (Sánchez-Valdes, Ortega-Ortiz, Ramos-de Valle, Medellín-Rodríguez, & GuedeaMiranda, 2009). Fig. 3 shows the ATR-FTIR spectra of gelatin and gelatineCW films in the range of 4000e650 cm 1. The addition of CW caused a

0.35

Gelatin Gelatin + 10% NC

0.30

0.25

Absorbance

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0.20

0.15

0.10

0.05

0.00 4000

3500

3000

2500

2000

1500

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Wavenumber (cm-1) Fig. 3. FTIR spectra of films CW0S and CW10S.

slight increase in the intensity of amide A (w3280 cm 1) band, and decreases in intensities of amide I (w1631 cm 1), amide II (w1542 cm 1) and amide III (w1238 cm 1) bands of gelatin. The increased intensity of amide A indicates that the addition of CW decreased gelatin hydration (Yakimets et al., 2005), because of competitive binding to water molecules, which can contribute to the antiplasticizing effect of CW evidenced by reduced elongation of nanocomposite films. The decreases in intensities of amides I, II and III may be attributed to a protein dilution effect from partial replacement of gelatin by cellulose whiskers (Núnez-Flores et al., 2013), and to amide-cellulose interactions causing conformational changes. The vibrational frequency of C]O band of amide I is dependent on the hydrogen bonding between amide units (Gaihre, Aryal, Barakat, & Kim, 2008), so the slight frequency upshift of amide I peaks in the nanocomposite film (from 1629.5 to 1631.5) indicates disruption of hydrogen bonding at carbonyl groups of gelatin (Núnez-Flores et al., 2013) by competitive binding of cellulose whiskers. The peak around 1035 cm 1 may be associated to interactions between hydroxyl groups of glycerol and the film components (Bergo & Sobral, 2007; Hoque, Benjakul, & Prodpan, 2011). The addition of CW resulted in a downshift (from 1034.5 to 1031.7 cm 1) in this peak, probably because of extra interactions between glycerol and the film structure (Bergo & Sobral, 2007). Some increases in peaks between 750 and 1000 cm 1 resulting from CW addition may be attributed to the presence of sulfonate groups from the sulfuric acid hydrolysis of cellulose (Morais et al., 2013), while the increased peak at 1160 cm 1 may represent the

Fig. 2. SEM images of films CW15 (A) and CW15S (B), presenting the effect of sonication on dispersion of cellulose whiskers.

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