Functional properties of gelatin-based films containing Yucca schidigera extract produced via casting, extrusion and blown extrusion processes: A preliminary study

Journal of Food Engineering 113 (2012) 33–40

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Functional properties of gelatin-based films containing Yucca schidigera extract produced via casting, extrusion and blown extrusion processes: A preliminary study Caroline Andreuccetti a, Rosemary A. Carvalho b, Tomás Galicia-García c, Fernando Martinez-Bustos c, Rubén González-Nuñez d, Carlos R.F. Grosso a,⇑ a

Department of Foods and Nutrition, Faculty of Food Engineering, University of Campinas, UNICAMP, 13083-862 Campinas, SP, Brazil Food Engineering Department, ZEA-FZEA, University of São Paulo, USP, 13635-900 Pirassununga, SP, Brazil CINVESTAV-Unidad Querétaro, Libramiento Norponiente 2000, Fracc. Real Juriquilla, Qro, 76001 Querétaro, Mexico d Department of Chemical Engineering, University of Guadalajara, Blvd. Gral. Marcelino García Barrazán 1451, 44430 Guadalajara, Jl, Mexico b c

a r t i c l e

i n f o

Article history: Received 30 November 2011 Received in revised form 19 March 2012 Accepted 18 May 2012 Available online 30 May 2012 Keywords: Biodegradable films Gelatin Yucca schidigera Surfactant Thermoplastic process Extrusion

a b s t r a c t Gelatin-based films containing both Yucca schidigera extract and low concentrations of glycerol (0.25–8.75 g per 100 g protein) were produced by extrusion (EF) and characterized in relation to their mechanical properties and moisture content. The formulations that resulted in either larger or smaller elongation values were used to produce films via both blown extrusion (EBF) and casting (CF) and were characterized with respect to their mechanical properties, water vapor permeability, moisture content, solubility, morphology and infrared spectroscopy. The elongation of the EF films was significantly higher than that of the CF and EBF films. The transversal section possessed a compact, homogeneous structure for all of the films studied. The solubility of the films (36–40%) did not differ significantly between the different processes evaluated. The EBF films demonstrated lower water vapor permeability (0.12 g mm m2 h1 kPa1) than the CF and EF films. The infrared spectra did not indicate any strong interactions between the added compounds. Thermoplastic processing of the gelatin films can significantly increase their elongation; however, a more detailed assessment and optimization of the extrusion conditions is necessary, along with the addition of partially hydrophobic compounds, such as surfactants. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction The production of edible or biodegradable films based on proteins can be accomplished using either a wet or dry process. The widely employed wet process, know as casting requires protein solubilization followed by a drying step to remove the solvent (Kester and Fennema, 1986; Debeaufort et al., 1998). However, casting is a discontinuous process. The dry process uses thermoplastic biopolymers associated with plasticizers and low levels of moisture (Hernández-Izquierdo and Krochta, 2008). Thermoplastic processes include casting via compression, injection, extrusion, blown extrusion and injection molding (Cuq et al., 1998; Verbeek and van den Berg, 2010). These processes usually result in a reorganization of the protein structure that favors a molecular alignment in the direction of flow (Hernández-Izquierdo and Krochta, 2008). During thermoplastic processes, both the mechanical energy and temperature contribute to the extensive denaturation, aggregation and crosslinking of the proteins. However, these techniques can be used for large-scale ⇑ Corresponding author. Tel.: +55 19 35214079; fax: +55 19 35214060. E-mail address: [email protected] (C.R.F. Grosso). 0260-8774/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jfoodeng.2012.05.031

production (Hernández-Izquierdo and Krochta, 2008; HernándezIzquierdo et al., 2008; Liu et al., 2005; Pommet et al., 2005). Recent reports have documented the characterization of protein-based films produced via thermoplastic processes, such as the extrusion of soy protein (Zhang et al., 2001), wheat gluten (Redl et al., 1999), whey protein (Hernández-Izquierdo et al., 2008), zein (Wang and Padua, 2003) and gelatin (Park et al., 2008; Krishna et al., 2012); the compression molding of collagen hydrolyzate (Haroun and El Toumy, 2010), myofibrillar protein (Cuq et al., 1997), soy protein (Cunningham et al., 2000), sunflower protein isolate (Oliarc et al., 2003), wheat gluten (Pommet et al., 2005), and whey protein (Sothornvit et al., 2003); the injection molding of soy protein (Huang et al., 1999) and the blown extrusion of zein (Wang and Padua, 2003; Oliviero et al., 2010). The denaturation and mobility of the protein chains is essential to successfully forming a film during the thermoplastic process, and the addition of plasticizers can improve the processability by favoring increased free volume and chain mobility (Verbeek and van den Berg, 2010). According to these authors, hydrophobic interactions are very important to the protein chain associations during the thermoplastic process. For this reason, amphiphilic plasticizers can more efficiently bind both the hydrophobic and

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C. Andreuccetti et al. / Journal of Food Engineering 113 (2012) 33–40

hydrophilic regions of the protein compared to hydrophilic plasticizers (di Gioia and Guilbert, 1999). Yucca schidigera is a plant of the Agavaceae family that grows in both México (Baja California) and the USA (Nevada, Arizona and California) (Piacente et al., 2004; Güçlü-Üstündag and Mazza, 2007), contains both steroidal saponins (10%) and phenolic compounds, possesses antioxidant activity (Oleszek et al., 2001; Piacente et al., 2004), and its extracts are permitted for use in the food industry in the US (under section 172.50, Natural Flavoring Substances and Natural Substances Used in Conjunction with Flavors, US Food and Drug Administration, 2011). Saponins are natural surfactants that contain glycosides of steroids or polycyclic terpenes (hydrophobic side) with a sugar chain (hydrophilic side) (Schenkel et al., 2007). The effects of Y. schidigera extract on the functional properties of gelatin-based films produced by casting were recently evaluated (Andreuccetti et al., 2011). Although the water vapor permeability of the film was significantly reduced relative to those not containing yucca extract, its mechanical properties, especially the % elongation, were not improved, thus producing films with low extensivity. As the thermoplastic process can more strongly modify both the protein structure and the functional properties of the resultant films than the casting technique, this study aimed to both produce and characterize gelatin-based films containing a Y. schidigera extract using the extrusion and blown extrusion processes. Additionally, the best formulation was used to produce and characterize films via the casting technique. 2. Materials and methods 2.1. Materials The following materials were used to create the films: type A pigskin gelatin (260 bloom) supplied by Gelita México (Lerma, State of México); natural Y. schidigera extract (yucca) supplied by Agroin Baja Agrointernacional, lot UBA-028 (Ensenada Baja California, México), as a surfactant; and glycerol as a plasticizer (CAS no. 56-81-5, J. T. Baker, Phillipsburg, NJ, USA). 2.2. Preparation of biodegradable films 2.2.1. Extrusion process Initially, the extrusion-processed films were formulated using gelatin and the yucca extract surfactant without a plasticizer. The thermoplastic processing of this blend was possible and generated homogeneous films. However, after 24 h of storage, the films lost moisture and became brittle. Therefore, it was necessary to include small quantities of a hydrophilic plasticizer (glycerol) in an attempt to minimize this problem. After adding glycerol, the EF was processed again and was visually homogeneous without granules, which indicates an efficient plasticization of the material. The EF remained manageable and flexible even after storage. The gelatin was blended with the glycerol and yucca extract in the quantities required for a factorial experiment (Table 1). Distilled water was added to obtain a 35% moisture level to allow for good flow through the extrusion system as determined by preliminary experiments. The samples were allowed to rest (5 ± 2 °C, 60 ± 2% relative humidity, RH) at least 24 h to allow for better moisture homogenization before being processed in the extruder. The size of the samples was adjusted using a sieve (20 mesh diameter). A laboratory single screw extruder (Cinvestav, Queretaro, México) with an L/D ratio of 20:1, screw compression ratio of 13:1, and 40 mm rectangular die was used. The barrel temperature

Table 1 Factors, variation levels, central composite design matrix with code and real variables and some functional properties for extruded films processed with yucca extract and glycerol. Code variables (real variables)

Functional properties

X1 (YC)1

TS (MPa)3

2

+1 (58) 1 (38) 1 (38) +1 (58) 0 (48) 0 (48) +1.414 (62.14) 1.414 (33.86) 0 (48) 0 (48) 0 (48)

X2 (GC)1 +1 (8.0) 1 (1.25) +1 (7.5) 1 (1.5) 1.414 (0.25) +1.414 (8.75) 0 (4.75) 0 (4.0) 0 (4.5) 0 (4.5) 0 (4.5)

4

24.3 ± 3.7 25.4 ± 0.9 22.8 ± 2.0 33.7 ± 11.7 33.8 ± 6.6 19.5 ± 1.4 21.8 ± 1.1 20.2 ± 1.6 25.2 ± 1.2 27.1 ± 5.3 20.7 ± 1.4

%E (%)3

MC (%)3

142.9 ± 16.7 75.6 ± 1.6 126.7 ± 3.1 80.9 ± 1.6 40.3 ± 4.1 157.8 ± 23.2 141.0 ± 5.9 114.2 ± 2.1 59.8 ± 0.6 124.4 ± 23.9 136.0 ± 8.9

16.9 ± 0.6 13.9 ± 1.7 15.9 ± 0.5 15.7 ± 0.7 14.2 ± 0.2 15.1 ± 1.2 15.8 ± 0.2 15.1 ± 0.1 16.2 ± 0.1 14.6 ± 1.4 12.8 ± 3.9

1 X1 = YC = Yucca concentration (g per 100 g protein); X2 = GC = Glycerol concentration (g per 100 g protein). 2 Values in brackets correspond to real variables. 3 TS: tensile strength, %E: elongation, and MC: moisture content. 4 Average ± standard deviation.

was 65 °C in the feeding zone and 100 °C in the intermediate zone, while the die temperature was 75 °C. These three zones were electrically heated independently and air-cooled. The feed screw was held at a constant 18 rpm, and the screw speed was 47.2 rpm. The temperature inside the heating zones was controlled using thermocouples. The variables studied were the yucca concentration (YC) and glycerol concentration (GC), and the analyzed responses were the mechanical properties (tensile strength, TS, and elongation, %E) and moisture content (MC). Both the coded and real values of the independent variables in the experimental design matrix, related to the levels 1 and +1, are shown in Table 1. All experiments were conducted randomly. 2.2.2. Blown extrusion process The gelatin, yucca extract and glycerol blend was prepared using the conditions described in Section 2.2.1. Prior to the blown extrusion, pellets were prepared using a laboratory single-screw extruder (Cinvestav, Querétaro, México) with an extruder barrel 380 mm long and 19 mm in diameter and a compression ratio of 1:1. A rectangular die with multiple orifices of 0.5 cm each was used. The barrel was equipped with electrical cartridge heaters and possessed three independently controlled heating and cooling zones controlled by thermocouples. The barrel temperatures in the feeding, intermediate and die zones were 30, 40, and 50 °C, respectively. The pellets were stored at 5 ± 2 °C and 60 ± 2% RH before processing by blown extrusion. A single-screw extruder (Haake Model Rheomex 254, Germany) coupled with a blown film device was used during the blown extrusion process to prepare tubular films. The technical specifications of the experimental setup were as follows: a screw diameter (D) of 19 mm, a screw length of 25 D, three heating zones (feeding 60 °C, intermediate 80 °C and compression 90 °C), an annular die with a diameter of 25 mm, a pin diameter of 24 mm and a gap width of 0–0.8 mm, and a heater capacity/zone of 1000 W. The other extrusion parameters were the screw speed (25 rpm), initial film radius (40 mm), both initial (0.5 mm) and final (0.18 mm) film thicknesses, nip rolls speed (35 rpm) and die temperature (105 °C). The thickness relation (TR), blow-up ratio (BUR, which indicates the amount of stretching in the hoop direction of the bubble), take-up ratio (TUR, which indicates the amount of stretching along the long axis of the bubble), and formation radius (FR, which indicates the balance of stretching, and thus the orientation, between the machine and transverse direction) were calculated according to the following equations as defined by Cantor (2006)

C. Andreuccetti et al. / Journal of Food Engineering 113 (2012) 33–40

35

TR ¼ die thickness=bubble thickness

ð1Þ

2.6. Water solubility

BUR ¼ Db=Dd

ð2Þ

The solubility of the films (2 cm discs) was determined according to the method described by Gontard et al. (1994) after 24 h of immersion in distilled water (50 ml) with mechanical stirring (Shaker Marconi-MA141, São Paulo, Brazil) at room temperature (25 ± 2 °C). The samples were then removed from the solution and dried in a forced air oven (105 °C for 24 h). The initial dry mass was determined from the sample moisture content. These determinations were performed in triplicate.

where Db is the bubble diameter, and Dd is the die diameter

TUR ¼ ðqAÞdie gap =ðqAÞnip rollers

ð3Þ

where q is the density (determined according Brandalero et al. (2010)), and A is the annular area

A ¼ pðR20  R2i Þ

ð4Þ

where R0 is the outer radius, and Ri is the inner radius of the annulus

FR ¼ TUR=BUR

ð5Þ

2.2.3. Casting method The gelatin was hydrated at room temperature (25 ± 2 °C) for 30 min and then solubilized at 55 °C for 15 min. Yucca extract and glycerol were mixed in distilled water (10 g 100 g of filmogenic solution) at room temperature. The solubilized gelatin (4%) was added to the surfactant/plasticizer solution and then blend homogenized at 10,000 rpm for 1 min using a homogenizer (UltraTurrax IKA, T18, Werke, Germany). The resulting homogenized solution was vacuum-filtered to remove air bubbles and the filmogenic solution was dispersed onto plexiglass plates (14 cm diameter) and kept at room temperature (25 ± 2 °C; 50–60% RH) to dry. 2.3. Film characterization, visual aspect and thickness The thickness of the films was determined using a Mytutoyo Corp. digital micrometer (Tokyo, Japan) using an average of 10 random measurements. Before characterization and after drying, the films were stored in desiccators (25 ± 2 °C, 50 ± 2% RH, Mg(NO3)26 H2O) for 7 days. Both visual and tactile analyses were conducted for all of the obtained films with the aim of only using homogeneous films with uniform color and without any phase separation, exudation, insoluble particles, and brittle zones. 2.4. Mechanical properties Because of the different thicknesses obtained with the processes used to produce the films, two methods for evaluating the mechanical properties were required, each using a texturometer, such as a TA-XT2 (TA Instruments, Newcastle, USA). For both the EF and EBF films, the mechanical properties were determined using the ASTM D638-00 method (ASTM, 2000); the initial separation distance and velocity were adjusted to 65 mm and 1 mm s1, respectively, using a 5 kg load cell. For the CF films, the TS and %E were determined using the ASTM D882-95 method (ASTM, 1995a); the initial separation distance and velocity were adjusted to 50 mm and 1 mm s1, respectively, and conditioned samples (25 mm  100 mm) were evaluated using a 5 kg load cell. All mechanical determinations were performed in triplicate. 2.5. Water vapor permeability The water vapor permeability (WVP, g mm m2 h1 kPa1) at 25 ± 2 °C was determined using the ASTM E96–95 method (ASTM, 1995b). The cells were filled with anhydrous calcium chloride (0% RH), covered with the conditioned films, sealed and placed in desiccators containing a saturated NaCl solution (75 ± 3% RH). The samples were weighed in triplicate five times each over a 48 h period, and the data were recorded in a graph of weight gain vs. time. The WVP was calculated according ASTM E96-95 method.

2.7. Scanning electron microscopy The films were conditioned in desiccators containing silica gel (25 °C) for a 7 day period and then fractured with liquid nitrogen. These samples were then coated with gold in a sputter coater, POLARON SC7620 (Ringmer, England), at 3–5 mA for 180 s, and the morphology the film surface and internal structures was observed using a scanning electron microscope, LEO 440i (Cambridge, England), at 5 kV. 2.8. Infrared spectroscopy The Fourier transform infrared spectroscopy (FTIR) analyses were conducted according to the procedure of Vicentini et al. (2005) using a Perkin Elmer Spectrum One spectrometer (Perkin Elmer, USA) equipped with a universal attenuated total reflectance (UATR) accessory at room temperature. The films were placed on the support and pressed with the measuring sensor. Ten scans were acquired for each sample over the spectral range of 400– 4000 cm1 with a 4 cm1 resolution. The data were analyzed using the FTIR Spectrum software (Perkin Elmer). 2.9. Experimental design and statistical analysis The effect of the yucca extract and plasticizer (glycerol) concentrations on the mechanical properties and moisture content of the extruded films (EF) were evaluated using an experimental design (22 + a) (Neto et al., 2002). The average values for the tensile strength, elongation, solubility and moisture of the EF, EBF and CF films were compared and their differences determined by the Tukey multiple test at the 95% confidence level using SAS for Windows Version 8.0 (Cary, North Carolina, USA). 3. Results and discussion 3.1. Extruded films The single screw extruder prototype used to process the gelatin-based films did not possess a calendering system that could adequately laminate the extruded material. As such, the traction of the processed material was performed manually, which resulted in an average thickness over all of the extruded films of 165 ± 10 lm (Table 1) and indicated good fluidization when passing through the extruder. Park et al. (2008) obtained thicknesses of 278 ± 19, 1059 ± 168 and 273 ± 10 lm for the films extruded from a gelatin base with added glycerol, sorbitol or a blend of these two plasticizers, respectively. The elevated thickness obtained for the film containing sorbitol was attributed to the low fluidity of the material inside the extruder when using this plasticizer. The barrel, intermediate and die zone temperatures, feeding velocity and screw speed were preliminarily adjusted to allow for complete plasticization after the die and a good flow through the barrel, which required temperatures of 100 °C in the intermediate zone and 75 °C in the die zone, respectively.

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The TS values (Table 1) varied between 19.5 and 33.8 MPa for formulations containing 48 g of yucca extract per 100 g of protein with 8.75 g of glycerol per 100 g of protein (HGLY) and 48 g of yucca extract per 100 g of protein with 0.25 g of glycerol per 100 g of protein (LGLY), respectively, which indicated that increasing the amount of glycerol decreased the TS. The effect of glycerol on the mechanical properties of the biodegradable films has already been thoroughly discussed in the literature. Increasing the amount of glycerol increases the elongation and reduces the tensile strength (Sothornvit and Krochta, 2001; Jongjareonrak et al., 2006). This effect was attributed to the modification of the tridimensional molecular organization of the gelatin, which decreases the intermolecular attractive forces between the protein chains and increases their free volume and mobility (Banker, 1966). The effect of the Y. schidigera extract addition on the functional properties of the gelatin-based films produced via casting has been previously evaluated (Andreuccetti et al., 2011). According to the authors, increasing the amount of yucca extract decreased the TS without significantly effecting the elongation, which produced films with low extensivity. The highest elongation value obtained was 157.8% for films using a formulation containing HGLY, and the smallest %E was 40.2% for films using the LGLY formulation (Table 1), higher values than those obtained for gelatin-based films containing yucca extract or both yucca extract and a hydrophobic plasticizer using casting (Andreuccetti et al., 2011, 2010). As preliminary experiments found that gelatin-based films or gelatin with yucca extract could be produced via the extrusion process but became brittle after 12 h of storage, the addition of low amounts of glycerol allowed for the control of moisture in the films after production and seems to reinforce the plasticizer effect of the yucca extract. The effect of temperature on the gelatin structure also needs to be considered. Rodríguez et al. (2006) added various surfactants (Tween 20, Span 80 and soy lecithin) to potato starch and glycerol-based films and observed a synergic effect between the glycerol and high concentration of surfactants used. These authors also observed that formulations containing glycerol with elevated surfactant concentrations resulted in films with low tensile strength and high elongation, which behaved like films with an elevated quantity of plasticizer in their polymeric chain. This apparently plastic film behavior at high levels of surfactants was only observed in the presence of glycerol (Rodríguez et al., 2006). The moisture content varied from 12.8% to 16.9% (Table 1). Formulations that produced films with both larger and smaller elongation values, which corresponded to those containing HGLY and LGLY, respectively, were used to produce films via extrusion followed by blown and casting. 3.2. Blown films Films produced via extrusion followed by blowing possessed a uniform surface without ruptures and with a characteristic color because of the presence of yucca extract (Fig. 1). No friction problems during bubble formation were observed. The calculated values in the extrusion-blowing process for the formulations containing HGLY were (i) a thickness relation of 2.7, which indicated a decrease of 2.7 times relative to the initial thickness; (ii) a BUR of 6.25; and (iii) a TUR of 0.65. An inferior stretching value in relation to the inflated air indicated that the directionality was greater in the transversal trajectory of the flow, and the formation radius of 0.10 indicated low bidirectionality during the film formation process. According to Cantor (2006), an FR  1 indicates a high bidirectionality in the film. The obtained relation of inflated air is an indicator that, when the thickness is increased, bubble formation is favored and the

Fig. 1. Blown extrusion of gelatin-based film with yucca extract and glycerol.

adhesiveness decreases throughout the cooling column. This differential was present because the beginning of bubble formation traction occurred manually along the column before reaching the roller handles. A limiting factor was observed during the process. Because of the sample adhesiveness, only a single layer was obtained with a thickness of approximately 0.5 mm. A decreased adhesiveness along the cooling column may be obtained by controlling the speed of the equipment and the internal volume of air, which increases the relationship between the initial and final film thickness (Cantor, 2006). During the processing of tubular films based on starch, glycerol and water, Thunwall et al. (2008) also reported adhesiveness problems with the formation of a single-layer of material. In an attempt to solve this problem, Thunwall et al. (2008) reduced the moisture content and glycerol concentration of the pellets. However, the formed film was fragile with bubble bursts, and the low glycerol concentration employed (15 parts glycerol per 100 parts dry starch) induced a significant increase in the torque required by the extruder. To avoid the adhesion of blown bubble layers in the present study, the material was cut before reaching the roller handles. After this modification, only a single layer was obtained. Additionally, the behaviors described by Thunwall et al. (2008) for films with low glycerol concentrations were not observed in this study. Here, smaller quantities of glycerol were used (a maximum of 8.75 parts glycerol per 100 parts gelatin) in association with the yucca extract, and the joint action of these compounds was still sufficient to promote the formation of blown films. Adjusting the film adhesiveness allows for a truly continuous process using roller handles while improving the bidirectionality (FR) of the films. The average thickness observed for the blown films (Table 2) varied based on the quantity of glycerol added to the mixture, which indicates either larger or smaller fluidity during the tubular film formation in the blown process. For the composition with the lowest glycerol concentration, the average thickness was 357 ± 15 lm, whereas the formulation containing HGLY yielded an average thickness of 166 ± 3 lm. In the present study, samples possessed an initial moisture content of 35%, which was adequate for the formation of gelatin-based

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C. Andreuccetti et al. / Journal of Food Engineering 113 (2012) 33–40 Table 2 Mechanical properties of films containing gelatin, yucca extract and glycerol, produced by extrusion, blown extrusion and casting method. Y C1

G C1

Method

Thickness (lm)

TS (MPa)2 b

%E (%)

48

0.25

Extrusion Blown extrusion Casting

167.6 ± 22.2d 357.0 ± 15.0a 55.5 ± 1.4e

33.9 ± 6.7 12.5 ± 1.8d 37.2 ± 0.2b

40.3 ± 4.1b 13.1 ± 3.1c 2.2 ± 0.1d

48

8.75

Extrusion Blown extrusion Casting

249.3 ± 23.7b 166.0 ± 3.0c 55.4 ± 3.2e

19.5 ± 1.4c 21.9 ± 1.2c 43.8 ± 1.0a

157.8 ± 3.2a 12.8 ± 2.7c 14.5 ± 1.9c

a,b 1 2

Average ± standard deviation. Different letters represent significant differences (p 6 0.05) between averages obtained through the Tukey test. YC = Yucca extract concentration (g per 100 g protein); GC = Glycerol concentration (g per 100 g protein). TS: tensile strength, %E: elongation.

blown films containing yucca extract and glycerol. According to Wang and Padua (2003), the moisture content and temperature are important factors in processing tubular films. These authors observed that the initial moisture content of the sample was essential for processing blowing zein resins into thin films. An elevated humidity content resulted in a material with a large number of water bubbles, which formed fragile and brittle films; however, a low moisture content resulted in a dry and rigid material that lacked plasticization. 3.3. Casting films The films produced via casting (CF) required a 12 h drying time under room conditions. This film was easily removed from the support surface, visually homogeneous without any insoluble particles or brittle areas, phase separated and possessing an average thickness of 55 ± 0.1 lm. Additional details about gelatin-based films containing yucca extract produced via casting have been previously reported (Andreuccetti et al., 2011). 3.4. Comparison between the functional properties of EF, EBF and CF 3.4.1. Mechanical properties Regardless of the plasticizer concentration, a higher %E was obtained via EF, which reached a maximum of 158% for the HGLY formulation versus 14.5% for CF and 12.8% for EBF, without a significant difference between the latter values (Table 2). The elongation of blown films based on zein was approximately 24% less than that obtained for the corresponding formulation processed using a screw extruder (Wang and Padua, 2003). These same authors reported that the method of the zein-based film formation greatly affected their mechanical properties because the rate of moisture evaporation promoted the different processes being employed. Although EBF films possessed elongation values under 15%, which was similar to those obtained for casting films, they were resistant and extensible enough to enable the production of tubular films. The highest TS was obtained via CF (43.8 MPa) rather than either EBF or EF when using HGLY (Table 2). The same behavior was observed by Park et al. (2008) while working with gelatin, sorbitol and glycerol-based films as well as Hernández-Izquierdo et al. (2008) while working with whey protein films where higher tensile strengths were obtained for casting films and higher elongation was obtained for extruded films. Park et al. (2008) obtained TS and %E values of 17 MPa and 216%, respectively, for extruded films and 60 MPa and 20%, respectively, for casting films using 20% glycerol. However, Hernández-Izquierdo et al. (2008) observed mean TS and %E values of approximately 3.5 MPa and 127%, respectively, for extruded films and 2.2 MPa and 57%, respectively, for casting films containing 45 to 55% glycerol. Importantly, the amount of glycerol added to the gelatin and yucca extract films in this study was lower (0.25–8.75%) than those used by these authors.

The differences in the mechanical properties because of the processing (extrusion, blown and casting) result from the different film formation mechanisms. During the extrusion process, protein molecules change structural orientation, and some degree of denaturation occurs, which favors a polypeptide chain alignment in the direction of the flow, improves the extensivity and generates a more flexible material (Hernández-Izquierdo et al., 2008). These property differences are also caused by the drastic conditions (high temperatures and pressure) applied to the material during the extrusion process, which modifies the protein structure (Ha and Padua, 2001; Sothornvit et al., 2007; Hernández-Izquierdo et al., 2008). The maximum temperature used in the present study was 100 °C in the shear zone, which could be sufficient to enable the formation of flexible, easily manageable films without brittle zones. Thermoplastic processing, whether by extrusion or blown extrusion, produced significantly thicker films than those obtained via casting (Table 2). A similar behavior was observed by Park et al. (2008). For EF films, the thickness increased with increasing glycerol content, which resulted in a reduced TS and increased %E. For the CF films, the plasticizer concentration did not significantly change the film thicknesses; however, a small increase in both the TS and %E was observed with increasing plasticizer concentrations. The thickness differences obtained for the EBF films indicated the effect of the plasticizer concentration on the formation of tubular films. The formulation containing the largest amount of glycerol yielded the lowest thickness, which suggests an improved stretching of the material during bubble formation. Films based on milk serum protein and containing glycerol showed an average thickness of 1310 ± 20 lm when processed using a twin screw extruder (Hernández-Izquierdo et al., 2008). These authors observed that the film thicknesses were not significantly affected by the glycerol content.

3.4.2. Scanning electronic microscopy The cross-sectional images obtained of the films produced with formulations that resulted in higher elongation values for EF, EBF and CF are shown in Fig. 2. The internal structures of the extruded, blown and casting films (Fig. 2D–F, respectively) were homogeneous, compact and similar, without any apparent micro pores. Park et al. (2008) compared gelatin-based films produced via either casting or extrusion and observed that films produced via casting possessed a more compact morphology. According to these authors, the more rapid water evaporation during extrusion relative to casting produced micro pores inside the films. Additionally, the surface morphologies of the films produced via extrusion and casting were homogeneous, continuous and similar (Fig. 2A and C). However, the surface images obtained for the EBF film (Fig. 2B) indicated some relieved sections, which can result from a structural rearrangement of the blown material during the cooling step.

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Fig. 2. SEM micrographs of the surface (A–C) and internal structures (D–F) of gelatin-based films containing 48 g yucca extract per 100 g protein plus 8.75 glycerol per 100 g protein produced by extrusion, blown extrusion and casting processes.

3.4.3. Water vapor permeability The water vapor permeability (Table 3) was only measured for the films produced from the formulation containing the highest glycerol concentration. From Table 3, it should be emphasized that the WVP of the EBF films was inferior to that obtained for both EF and CF films. According to Hernández-Izquierdo and Krochta (2008), the structural differences in the protein matrix caused by processing via either extrusion or extrusion followed by blowing produce a more ordered matrix than that of casting films, which increases the barrier to water vapor. For CF films, the protein chains would be arranged in a more chaotic manner with larger distances between them, which would allow for better water vapor diffusion through the filmogenic matrix and, therefore, promote film permeability. However, Park et al. (2008) reported higher WVPs for the

extruded films (based on gelatin, glycerol and sorbitol) rather than the casting films. According to these authors, films obtained via the extrusion process possessed micro voids, whereas casting films possessed a more compact structure with less interstitial spaces between the gelatin molecules, thereby lowering their WVP. McHugh et al. (1993) observed that the WVP increased as the thickness of the gelatin films increased. However, in the present study, the films produced via extrusion blown possessed both higher thicknesses and lower WVP values than CF films. In this case, the inclusion of a surfactant with hydrophobic characteristics modified the behavior observed by McHugh et al. (1993). The addition of yucca extract to gelatin-based films has been stated as an efficient means to reduce the WVP (Andreuccetti et al., 2010, 2011).

Table 3 Solubility, moisture content and water vapor permeability for the gelatin-based films, yucca extract and glycerol, produced by extrusion, blown extrusion and casting method. Y C1

a,b

G C1

Method

SOL (%)2

MC (%)2 a

WVP (g mm m2 h1 kPa1)2 b

48

0.25

Extrusion Blown extrusion Casting

37.1 ± 0.8 27.1 ± 0.6b 39.4 ± 2.4a

13.6 ± 1.0 16.6 ± 0.2a 9.2 ± 0.8c

– – –

48

8.75

Extrusion Blown extrusion Casting

39.8 ± 1.6a 36.4 ± 2.4a 39.5 ± 5.6a

16.5 ± 0.7a 14.1 ± 1.4b 8.3 ± 0.4c

0.16 ± 0.05ab 0.12 ± 0.02b 0.16 ± 0.00a

Average ± Standard deviation. Different letters represent significant differences (p 6 0.05) between averages obtained through the Tukey test. YC = yucca extract concentration (g per 100 g protein); GC = glycerol concentration (g per 100 g protein). 2 SOL: solubility, MC: moisture content, WVP: water vapor permeability. 1

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39

Fig. 3. FT-IR spectrum of gelatin-based films produced by extrusion (EF), blown extrusion (EBF) and casting (CF) processes containing 48 g yucca extract per 100 g protein plus 8.75 g glycerol per 100 g protein.

3.4.4. Solubility When the different processes were compared (EF, EBF and CF), it was found (Table 3) that the film solubility did not differ significantly (p > 0.05), regardless of the formulation and production method utilized, with the exception of the EBF film obtained using the lowest glycerol concentration, which possessed a significantly lower solubility (27%) than the other treatments. The film solubilities observed in this study, regardless of the production method, were comparable to those obtained using gelatin-based films produced via casting, which range from 25% (Carvalho et al., 2004) to 39.9% (Gómez-Estaca et al., 2009). In this study, the addition of glycerol had no significant effect on the solubility, possibly because of the low plasticizer concentrations used (0.25–8.75 g per 100 g protein). However, Sothornvit et al. (2003) observed that increasing the glycerol concentration in modeled compression films based on whey protein increased the solubility (30%, 36% and 49% solubility when 30%, 40% and 50% of glycerol were added, respectively). 3.4.5. Infrared spectroscopy The FTIR spectra of the films formed via extrusion, blown extrusion and casting were similar (Fig. 3), and the absorbed wavelengths did not change significantly, which indicates that the different processing methods did not favor the formation of additional interactions between the used compounds (protein, surfactant and plasticizer). The spectra obtained for the EF, EBF and CF films possessed peaks characteristic of gelatin (Hashim et al., 2010; Haroun and El Toumy, 2010) and the yucca extract (Andreuccetti et al., 2010) because of the presence of resveratrol and other compounds in the yuaccaol surfactants (Piacente et al., 2004). The gelatin films had peak absorption wavelengths between 3600 and 3500 cm1, which can be observed in their spectra (EF, EBF and CF), and primarily correspond to the OH stretching vibrations of absorbed water molecules resulting from the hygroscopic nature of this type of film (Yakimets et al., 2005). The absorbed wavelengths at approximately 1630–1660 cm1 correspond to random coils and that at 1660 cm1 corresponds to a triple helix with contributions from both a-helix and b-turns. Components of the amide absorp-

tion at 1690 cm1 were assigned to aggregated helices of collagen-like peptides (Prystupa and Donald, 1996). However, it was observed that the characteristic peaks differed in their absorbance intensities, especially for the spectra obtained from the CF film, which was significantly less intense than that obtained for the EF and EBF films. The peak intensities can indicate the amount of free water in the system, which is consistent with the significantly lower moisture content of the CF film than the EF and EBF films, as shown in Table 3. Pushpadass et al. (2009) reported that the height and area of absorption peaks with, for example, wavelengths of approximately 3200–3300 cm1, increased with increasing moisture or plasticizer contents, which increases the amount of OH groups in the resulting films. The shift of the CO group band to lower frequencies indicates the possibility of forming more stable hydrogen bonds between CO and the plasticizer (Ma et al., 2006); however, for the gelatin films, the wavelengths of the yucca extract and glycerol in this region were similar with only minor modifications, which is not indicative of either increased or decreased interactions. 4. Conclusions This study demonstrated that the thermoplastic processing of gelatin-based films containing yucca extract and small quantities of glycerol using a single screw extruder was possible. The formulation that produced the most elongation during the extrusion process was also sufficiently resistant and extensible to form tubular films via the blown technique, which provided a preferential orientation transversal to the flow with low rates of bidirectionality. For the extruded films, the yucca extract and glycerol concentrations did not significantly affect either the tensile strength or moisture content, whereas the glycerol concentration significantly affected the elongation values. Because of the different processing methods employed in forming these gelatin-based films, the extruded materials possessed increased elongation and reduced rupture tension relative to films obtained via casting. Regardless of the production method used to form the films, their morphology was similar, with compact and homogenous inner structures that lacked micro pores. The evaluation of the infrared spectra did not indicate a

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