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Film forming solutions based on gelatin and poly(vinyl alcohol) blends: Thermal and rheological characterizations
Journal of Food Engineering 95 (2009) 588–596
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Film forming solutions based on gelatin and poly(vinyl alcohol) blends: Thermal and rheological characterizations Izabel C.F. Moraes a, Rosemary A. Carvalho a, Ana Mônica Q.B. Bittante a, Javier Solorza-Feria b, Paulo J.A. Sobral a,* a b
Department of Food Engineering, FZEA, University of São Paulo, 13635-900 Pirassununga/SP, Brazil Centro de Desarrollo de Productos Bióticos del IPN, Carr. Yautepec-Jojutla, Km 6, calle CEPROBI No. 8, Col. San Isidro, C.P. 62731 Yautepec, Morelos, Mexico
a r t i c l e
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Article history: Received 6 March 2009 Received in revised form 17 May 2009 Accepted 14 June 2009 Available online 21 June 2009 Keywords: Viscoelasticity Viscosity Gel Helix-coil transition Differential scanning microcalorimetry Glycerol
a b s t r a c t The objective of this work was to study the rheological and thermal properties of film forming solutions (FFS) based on blends of gelatin and poly(vinyl alcohol) (PVA). The effect of the PVA concentration and plasticizer presence on the flow behavior, and viscoelastic and thermal properties of FFS was studied by steady-shear flow and oscillatory experiments, and also, by microcalorimetry. The FFS presented Newtonian behavior at 30 °C, and the viscosity was not affected neither by the PVA concentration nor by the plasticizer. All FFS presented a phase transition during tests applying temperature scanning. It was verified that the PVA affected the viscoelastic properties of FFS by dilution of gelatin. This behavior was confirmed by microcalorimetric analysis. The behaviors of the storage (G0 ) and loss (G00 ) moduli as a function of frequency of FFS obtained at 5 °C were typical of physical gels; with the G0 higher than the G00 . The strength of the gels was affected by the PVA concentration. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Packages made with synthetic plastic films display overall excellent functional properties, but usually they are non-biodegradable, and therefore lead to environmental pollution, which pose serious ecological problems (Tharanathan, 2003). Thus, researches on development of biodegradable films have increased in the last years. After all, biodegradability is not only a functional requirement but also an important environmental attribute (Tharanathan, 2003). Several raw materials may be used to obtain biodegradable films (Tharanathan, 2003), being outstanding the biopolymers from renewable sources such as gelatin (Arvanitoyannis, 2002), and also synthetic polymers, as the poly(vinyl alcohol) (PVA) (Matsumura et al., 1999). More recently, biodegradable films have been developed using mixtures of gelatin and PVA (Chiellini et al., 2001a,b,c). Gelatin is a protein of animal origin, resulting from the acid or basic hydrolysis of collagen coming from bones, bovine and porcine skins, and from connective tissues (Gennadios et al., 1994; Arvanitoyannis, 2002). This macromolecule has been used in films production, most probably due to its outstanding filmogenic properties and its high production volumes at competitive prices (Sobral et al., * Corresponding author. Tel.: +55 19 35654192; fax: +55 19 35654114. E-mail address: [email protected] (P.J.A. Sobral). 0260-8774/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2009.06.023
2001). Gelatin has the capacity to form physical gels, i.e., thermoreversible gels. At a molecular level, gelatin gel formation involves a structural re-arrangement of the protein, implying the change from a disordered stage to a more ordered one, formed by triple helix structures (Slade and Levine, 1987; Arvanitoyannis, 2002). The poly(vinyl alcohol) (PVA) is produced from poly(vinyl acetate), by hydrolysis of the functional acetate groups (Yamaura and Naioth, 2002; Sudhamani et al., 2003). The PVA is a synthetic polymer, water soluble, non-toxic, which has been used in various materials in the medical and biomedical areas (Yamaura and Naioth, 2002). It also has excellent film forming properties (Sudhamani et al., 2003). Biodegradable films from gelatin and PVA have been developed by the wet method, that is to say, by casting and dehydration of a film forming solution (FFS) (Chiellini et al., 2001a,b,c; Maria et al., 2008; Mendieta-Taboada et al., 2008; Silva et al., 2008; Carvalho et al., 2009). According to Cuq et al. (1995) and Peressini et al. (2003), the knowledge of the FFS rheological properties by stationary and dynamic tests, is important for the design and processing of films by casting. It may be found in the specialized literature that diverse studies on the rheological behavior, including the viscoelasticity, have been carried out with relatively highly viscous FFS (Cuq et al., 1995; Barreto et al., 2003; Peressini et al., 2003). However, few studies have been carried out about the characterization of low viscosity FFS, produced usually with low concentration of macromolecules (Peressini et al., 2003; Gómez-Giullén et al.,
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2007). Within this context, the purpose of this work was to study the rheological and thermal properties of relatively diluted film forming solutions, based on blends of gelatin and PVA. The main interest was to verify the effect of the PVA concentration and plasticizer presence on the flow behavior, and viscoelastic and thermal properties of FFS.
2. Materials and methods
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2.5. Dynamic tests For the dynamic tests, the extent of the linear viscoelastic region was determined by performing a strain (0–20%) sweep in FFS both as a gel (5 °C) and as a sol (30 °C), in all cases at 1 Hz of frequency. Thus, according to the results of these tests, the strain value used for all following dynamic tests was fixed at 4%, within the linear viscoelastic domain. The viscoelastic parameters determined in these trials were the storage or elastic modulus (G0 ) and the loss or viscous modulus (G00 ).
2.1. Materials (a) Temperature sweep tests The macromolecules used for film forming solutions (FFS) production were: pigskin gelatin (bloom 242–248; molecular weigh 5.2 104 Da; moisture content = 9.3%) supplied by Gelita South America (São Paulo, Brazil), and poly(vinyl alcohol) with a degree of hydrolysis (DH) of 91.8%, molecular weight (Mn, furnished by supplier) of 85–124 kDa and viscosity of 17.6 cp (CevolÒ418) supplied by Celanese (Dallas, USA). Glycerol (Synth) was used as plasticizer. 2.2. Sample preparation The film forming solutions (FFS) were produced from a mixture of gelatin (solution A) and PVA (solution B) solutions. The solution A was prepared as follows: gelatin (different concentrations) was hydrated for 30 min, and then dissolved at 55 °C (Sobral et al., 2001) using a thermostatic bath (Marconi, Model TE 184). When necessary, glycerol was added and the solution was held at 55 °C for further 15 min. To prepare the solution B, the PVA (different concentrations) was first homogenized in distilled water and then dissolved at 95 °C (Chiellini et al., 2001a) with magnetic stirring (Tecnal-TE085). Solutions A and B were then mixed and homogenized for 15 min at room temperature with magnetic stirring, so as to produce FFS with 2 g macromolecules/100 g FFS with different proportions of PVA and gelatin (0, 10, 20, 30, 40 and 50 g PVA/100 g macromolecules, corresponding to 0:100; 10:90; 20:80; 30:70; 40:60 and 50:50 of PVA:gelatin, respectively), and plasticizer concentrations of 0 and 25 g glycerol/100 g macromolecules. More details on that preparation can be observed on previous works (Maria et al., 2008; Mendieta-Taboada et al., 2008). These FFS were immediately analyzed to determine its rheological behavior by steady-shear tests, and dynamic tests, with temperature and frequency sweeps. Also, the phase properties of FFS were determined by differential scanning microcalorimetry (microDSC). All tests in this study were performed at least in triplicate. 2.3. Rheological measurements Rheological tests were carried out in a rheometer (AR2000 Advanced Rheometer; TA Instruments, New Castle, DE, USA) using a cone and plate sensor geometry (cone angle 4°, 60 mm diameter). This equipment controlled the temperature with a peltier system. The results were analyzed using the software Rheology Advantage Data Analysis V.5.3.1 (TA Instruments). Before undertaking any rheological tests, all samples remained at rest for at least 5 min between the upper cone and plate geometry and the lower fixed plate, to allow the relaxation of stresses induced during sample loading (Peressini et al., 2003). To avoid water losses due to evaporation, the measurement system was coated with a solvent trap accessory.
Firstly, the FFS was heated up to 50 °C without a predetermined heating rate program, beginning the tests when that measurement temperature was attained. The essays were actually divided in two parts, a test was run while the sample was cooled down from 50 to 5 °C, remaining at this latter temperature for 5 min., and then another test was run while heating up the sample to 50 °C, always at a scan rate of 1 °C/min. From the results of these tests, the G0 and G00 values as a function of temperature were used to determine the following parameters: gelling (Tsol–gel) and melting (Tgel–sol) temperatures, calculated as the temperature where G0 changed drastically as an inflexion point; and the viscoelastic moduli (G0 and G00 ) of the gel, calculated at 5 °C (Gómez-Giullén et al., 2007). (b) Frequency sweep tests Some tests were carried out in the sol domain, but the results (G0 values) were very low, i.e., beyond the limit of sensitivity of the equipment. Thus, it was decided to run the frequency sweep tests at 5 °C, solely in the gel domain of samples. This temperature was attained by cooling down the FFS at 1 °C/min. Then, samples were subjected to sweep tests from 0.1 to 10 Hz (Van den Bulcke et al., 2000; Peressini et al., 2003). In these tests, the results were presented as G0 and G00 as a function of frequency. 2.6. Thermal analysis Thermal analyses were carried out using a differential scanning microcalorimeter (nanoDSC III, CSC 6300, Calorimetry Sciences Corporation, UT, USA). To use this technique, the FFS had to be diluted (101) in distilled water because of the equipment sensitivity (1 lcal.). This solution was then added into the measurement cell using a micropipette. Distilled water was added to the reference cell. These cells were pressurized at 3 atm to avoid water evaporation during heating. The raw data were analyzed using the software Cpconvert v.2.1.1, Cpcalc Analysis Tools (CSC – Calorimetry Sciences Corporation). Before starting these analyses, the cells were cooled down to 2 °C without any control of the cooling rate, remaining at that temperature (isothermal) for 10 min. Then, the cells were subjected to the following analyses program, always at 1 °C/min: heating up from 2 to 80 °C (scan 1), staying isothermally at 80 °C for 10 min, cooling down from 80 to 2 °C (scan 2), remaining at the latter temperature for 10 min, and then heating up again from 2 to 80 °C (scan 3). The phase transition temperature was estimated as the peak temperature of the endothermal phenomenon, and the phase transition enthalpy was calculated as the area under this same peak (Sobral and Habitante, 2001; Habitante et al., 2008).
2.4. Stationary state tests 2.7. Statistical analyses Steady-shear measurements were performed in an extended shear rate range (0–200 s1), at 30 °C. According to previous tests, at this temperature the FFS was always in the sol domain.
Statistical analyses were carried out using the SAS program (Version 9.1, SAS Institute Inc., Cary, NC, USA) and in case of signif-
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icance, the differences between means were determined by the Duncan multiple test (p < 0.05). 3. Results and discussion 3.1. Flow behavior All results of the steady state tests showed similar behavior; the relationship between the shear stress and shear rate was linear. Examples of these profiles are shown in Fig. 1. The average viscosity value of FFS was about 7.1 ± 0.1 mPa s, irrespective of the concentrations of the PVA or glycerol. This Newtonian behavior appeared certainly because the FFS were in fact diluted solutions (2%). In these cases, the behavior is mainly linked to the macromolecules–water interactions rather than to the macromolecules–macromolecules ones (Barreto et al., 2003). The organization of the polypeptides (gelatin) in the already known triple helix, as well as the associations of these organized sequences, could take place, depending on factors such as gelatin chemical structure, concentration and temperature, affecting the FFS rheological behavior. This last tendency of macromolecules interaction has been observed in more concentrated systems, with a consequent non-Newtonian behavior (Xiong and Blanchard, 1994; Cuq et al., 1995; Peressini et al., 2003; Sato et al., 2008). Fitzsimons et al. (2008) determined flow curves for both unpolymerized and polymerized whey protein isolate (6% WPI, pH 7), alone and mixed with 1% and 2% of type B gelatin and observed Newtonian behavior, with viscosity values around 2 mPa s. When these authors mixed the WPI with 3% or 4% of gelatin, some slight shear-thinning behavior was noticed. Barreto et al. (2003) studied sodium caseinate FFS as a function of biopolymer concentration, temperature and plasticizer content and also observed Newtonian behavior; consistent with this work, with no effect of the plasticizer (sorbitol) concentration on the viscosity of diluted FFS. The leveling of irregularities in liquid coating (or in FFS casting), depends mainly, on their rheological properties (Peressini et al., 2003). According to Cuq et al. (1995), to cast a high viscosity FFS is very difficulty and needs a dispersing machine (spreader). Nevertheless, the FFS studied in this work had low viscosity values, which readily allowed casting these solutions by free flow (Maria et al., 2008; Mendieta-Taboada et al., 2008; Silva et al., 2008; Carvalho et al., 2009).
Fig. 1. Examples of flow curves determined at 30 °C, of film forming solutions with 0% of PVA with () and without glycerol (j), and with 50% of PVA without glycerol (h).
3.2. Scanning temperature in dynamic rheological tests Similar rheological profiles were observed in all temperature sweep results (Figs. 2 and 3); within the range studied, both moduli (G0 and G00 ) had higher values at low than at relatively high temperatures. Between these two domains, an inflexion was observed in both moduli. These behaviors are typical of physical gels presenting a sol–gel (cooling, Tsol–gel) and/or a gel–sol (heating, Tgel– sol) transition, such as gelatin. Moreover, as expected, the viscous modulus (G00 ) was higher than the elastic modulus (G0 ) over the sol domain temperature and when the systems showed a gel character, it was also observed that the stiffness (elastic character) increased with gelatin concentration in the blend (Figs. 2 and 3). Similar profiles have been observed by several authors in gelatin based systems (Van den Bulcke et al, 2000; Cho et al., 2005; Gómez-Giullén et al., 2007). Overall, a sol–gel or a gel–sol transition of proteins is a multistep thermodynamic process that involves a three-dimensional network structure formation (Xiong and Blanchard, 1994). That is to say, the gelation of gelatin is the result of intermolecular interactions among protein molecules usually involving hydrogenbonding, electrostatic and hydrophobic interactions (Gennadios et al., 1994). Gelatin is then, an entangled polymer network that, depending upon the specific temperature and time of application, is interspersed with certain number of crosslinks that affects its rheological, and mechanical properties. The temperatures of the sol–gel and gel–sol transitions, fitted as the temperature where the peak of the curve of the first derivative of G0 occurred, were affected by the PVA concentration, without a notable effect due to glycerol (Table 1). Peressini et al. (2003), working with methylcellulose (MC) and glycerol solutions, also observed that the MC concentration had an effect much more significant on the viscoelastic properties of these solutions, than the glycerol concentration. Overall, the increase in the PVA concentration in the blend, caused an intense reduction (47%) in the Tsol–gel, which decreased from around 15 °C to about 8 °C, whereas the Tgel–sol decreased (14%) from around 29 °C to about 25 °C, irrespective of the presence of glycerol in the FFS. In consequence, an increase in the hysteresis was observed, this meant that as the PVA concentration in the blend increased, the difference between Tgel–sol and Tsol–gel also increased (Table 1). These behaviors may well be associated with the processes involved in the coil-helix/helix-coil transition, i.e., the gelling of the solution and the melting of the gel. The gelatin triple helix formation (coil-helix transition) is a slow process, which continues almost indefinitely, while the dissociation (helix-coil transition) of the triple helix is an equilibrium process linked with the transition temperature (Fitzsimons et al., 2008). Cho et al. (2005) have performed dynamic rheological studies, by running temperature sweeps in dispersions with 6.67% of gelatin from pig, and determined higher Tgel–sol (36.5 °C) and Tsol–gel (25.6 °C) values than those found in this work with gelatin without PVA (Table 1). Even though these authors have used a different heating rate (0.5 °C/min), their results could possibly be because they have worked with more concentrated systems. However, it has to be noted that the hysteresis (11 °C) found by Cho et al. (2005) was similar to the one observed in this work (13 °C). Moreover, Cheow et al. (2007), who worked with 6.67% bovine gelatin solutions, determined intermediate values of Tsol–gel (19.6 °C) and Tgel–sol (28.8 °C). The effect of the PVA concentration on the transition temperature may be explained from the results of the viscoelasticity rheological tests with pure PVA solutions (Fig. 2). It was seen that this polymer practically did not show elastic behavior (G0 ? 0) and also, it did not display any phase transition within the range of temperatures studied. This suggests that probably the effect of
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Fig. 2. Storage (G0 ) and loss (G00 ) moduli of FFS based on blends of gelatin and PVA (CevolÒ418) without glycerol, as a function of temperature. (A and C) cooling; (B and D) heating, both at 1 °C/min.
Fig. 3. Storage (G0 ) and loss (G00 ) moduli of FFS based on blends of gelatin and PVA (CevolÒ418) with glycerol, as a function of temperature. (A and C) cooling; (B and D) heating, both at 1 °C/min.
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Table 1 Transition temperatures Tsol–gel; Tgel–sol and DT (hysteresis) of film forming solutions based on gelatin and PVA (CevolÒ418) with 0 and 25 g of glycerol/100 g of macromolecules.a Concentration of PVA (g/100 g)b
0 10 20 30 40 50 a b
With glycerol
Without glycerol
Tsol–gel (°C)
Tgel–sol (°C)
DT (°C)
Tsol–gel (°C)
Tgel–sol (°C)
DT (°C)
15.1 ± 0.3a 15.1 ± 0.4a 13.8 ± 0.3b 12.7 ± 1.0c 10.6 ± 0.5d 8.4 ± 0.5e
28.5 ± 0.3a 28.3 ± 0.1a 27.8 ± 0.2b 27.1 ± 0.4c 26.6 ± 0.6d 24.7 ± 0.1e
13.4 ± 0.2c 13.2 ± 0.4c 14.1 ± 0.4b 14.4 ± 0.9b 16.0 ± 0.6a 16.3 ± 0.4a
15.3 ± 0.2a 14.4 ± 0.2b 13.8 ± 0.4c 12.7 ± 0.1d 10.8 ± 0.2e 8.5 ± 0.4f
28.6 ± 0.3a 28.1 ± 0.3b 27.8 ± 0.3b.c 27.5 ± 0.2c 26.4 ± 0.4d 25.3 ± 0.4e
13.3 ± 0.2e 13.7 ± 0.2d 14.0 ± 0.4d 14.8 ± 0.2c 15.6 ± 0.4b 16.8 ± 0.3a
Different letters in the same column indicate significant differences (p < 0.05) among the average values, by application of the Duncan test using the SAS program. g PVA/100 g macromolecules.
the PVA on gelatin gelation, could have been nothing else but a consequence of the gelatin dilution in the blend, and thus, interfering with the continuity of the gel that this protein forms, by obstructing the conformational transition of gelatin molecules from random coils, to triple helical structures which joins several chains. Previous authors found that the gelatin gelation temperature was highly dependent on this protein concentration in solution, and in the present case, therefore, on the PVA concentration in the blend (Gómez-Giullén et al., 2007). With the reduction of the gelatin concentration in solution, the intermolecular interactions during the ‘‘helix” structure formation became less probable (Van den Bulcke et al., 2000). However, as previously mentioned, the occurrence of certain type of interaction between the PVA and gelatin, which could in consequence avoid gelatin junctions formation and therefore, that of the gelatin network cannot be disregarded. Van den Bulcke et al. (2000) studied the gelation of gelatin reticulated with meta-acrylamide, with different degrees of reticulation, and obtained results apparently similar to those presented in Figs. 2 and 3. The increase of the reticulation degree caused a decrease of the Tgel–sol, similar trend to that observed in this work (Table 1). Furthermore, these authors verified that the reticulation degree of the gelatins also affected the gels rigidity (stiffness), i.e., the values of the storage modulus (G0 ) of the gels at temperatures lower than the Tgel–sol, decreased as the gelatin reticulation degree increased. Similar results respect to the effect of the PVA concentration in the blend were obtained in this work: in all cases studied, an increase in the PVA concentration caused a decrease in the G0 values determined at 5 °C (Table 2). According to GómezGiullén et al. (2007), the rigidity of the gelatin gel is also highly dependent of this protein concentration in solution. Thus, it may be considered that, with the increase of the PVA concentration in the FFS, there were less intermolecular associations between proteins, and consequently, a weaker gel was formed. The presence of the plasticizer (glycerol) in the FFS did not affect neither the transition temperatures (Table 1) nor the G0 values determined at 5 °C (Table 2). The glycerol is a low molecular weight molecule, that when added to polymeric materials in relatively high concentrations (e.g., 10–40 g/100 g polymer), modifies
the tridimensional organization of the polymeric matrix, decreasing the intermolecular attraction forces and consequently, increasing the free volume and the mobility of the polymeric chain. This is done via hydrogen bonds between its hydroxyl groups and the polar functional groups of some amino acid residues (Gennadios et al., 1994; Cuq et al., 1995; Sobral et al., 2001). However, in the FFS production of the present work, the plasticizer concentration was very low; about 0.5% (g glycerol/100 g of FFS). Thus, the polar functional groups of the gelatin amino acids should have interacted firstly with water molecules, which were in greater numbers than those of glycerol. The production of films by casting implies, initially, the FFS manufacture, where the biopolymer is found in the form of sol. After the application of the FFS on a support material and then subjecting the FFS to drying, film formation takes place. In an intermediate stage, especially in the case of films made from gelatin, the film will necessarily pass by a gel formation, with a semi-rigid tridimensional matrix constitution, which will involve and immobilize the plasticizer and the solvent (water). Then, with the water evaporation by dehydration, the plasticizer will become more concentrated. Thus, the formation of the biopolymeric matrix will take place via electrostatic, hydrophobic, and Van der Walls interactions, as well as hydrogen bonds among adjacent biopolymer chains and between these and the plasticizer (Cuq et al., 1995). Effectively, the transition (Tsol–gel 6 15.3 °C; Tgel–sol 6 28.6 °C) temperatures of the studied FFS (Table 1) were below the usual drying temperature (30 °C) (Maria et al., 2008; Mendieta-Taboada et al., 2008). Thus, in that condition, it might be considered that the FFS could not be a real gel. But, in fact, the gel formation could have occurred during the application/drying of the FFS, either because the air wet bulb temperature was lower than the respective dry bulb temperature; or because a material concentration occurred during the drying process, which implied the increase of the transition temperatures, as observed by Menegalli et al. (1999). 3.3. Scanning temperature in microcalorimetry To supplement the temperature sweep dynamic rheological tests, microcalorimetric analyses were undertaken to the same
Table 2 Storage modulus (G0 ) of gels based on gelatin and PVA (2 g of macromolecules/100 g of FFS), with 0 and 25 g of glycerol/100 g of macromolecules, at 5 °C.a Concentration of PVA (g/100 g)b
0 10 20 30 40 50 a b
With glycerol
Without glycerol
Cooling (Pa)
Heating (Pa)
Cooling (Pa)
Heating (Pa)
198.1 ± 12.8a 131.6 ± 1.5b 84.8 ± 17.9c 19.4 ± 3.5d 12.0 ± 1.2d 2.2 ± 0.4e
296.5 ± 5.2a 216.4 ± 1.8b 145.1 ± 23.6c 42.5 ± 6.8d 24.8 ± 3.5e 8.0 ± 1.0f
206.0 ± 13.6a 132.7 ± 4.3b 81.8 ± 6.2c 38.5 ± 3.3d 12.8 ± 0.4e 2.3 ± 0.2f
305.9 ± 5.9a 212.2 ± 12.4b 136.9 ± 7.6c 71.0 ± 8.4d 27.8 ± 2.0e 7.7 ± 1.0f
Different letters in the same column indicate significant differences (p < 0.05) among the average values, by application of the Duncan test using the SAS program. g PVA/100 g macromolecules.
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previously described samples, diluted ten times due to equipment sensibility. In these analyses, the transition temperature was observed as a peak in the specific heat (Cp) variation as a function
of temperature (Fig. 4), in the heating thermograms. These endothermal peaks could be assigned to the gelatin triple helix dissociation, the so-called gel melting. It is interesting to note that the Cp
Fig. 4. Specific heat of diluted film forming solutions based on blends of gelatin and PVA, with (A and B) and without (C and D) glycerol and with different concentrations of PVA (indicated in the plotts): B and D – 1st scan; A and C – 3rd scan, obtained by microcalorimetry. Heating rate: 1 °C/min.
Table 3 Transition temperature (Tgel–sol) of film forming solutions based on gelatin and PVA (CevolÒ418) determined by microcalorimetry.a Concentration of PVA (g/100 g)b
With glycerol Tgel–sol (°C) (1° scan)
Tgel–sol (°C) (3° scan)
Tgel–sol (°C) (1° scan)
Tgel–sol (°C) (3° scan)
0 10 20 30 40 50
26.8 ± 0.5a 26.6 ± 0.5a 28.4 ± 3.0a 26.8 ± 0.4a 28.2 ± 2.7a 26.9 ± 0.3a
26.4 ± 0.1a 26.2 ± 0.2a 26.4 ± 0.0a 26.3 ± 0.1a 26.3 ± 0.1a 26.4 ± 0.3a
26.9 ± 0.4a 27.8 ± 2.5a 27.0 ± 0.1a 28.6 ± 2.9a 27.1 ± 0.5a 30.1 ± 2.7a
26.7 ± 0.2a,b 26.7 ± 0.3a,b 26.9 ± 0.1a 26.4 ± 0.4b 26.7 ± 0.1a,b 26.8 ± 0.2a
a b
Without glycerol
Different letters in the same column indicate significant differences (p < 0.05) among the average values, by application of the Duncan test using the SAS program. g PVA/100 g macromolecules.
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Fig. 5. Enthalpy (DH) of gel–sol transition as a function of PVA concentration (CPVA) in the blend of the film forming solutions of gelatin and PVA. (A) 1st scan; (B) 3rd scan.
peaks were almost independent of the PVA concentrations in the blends. This behavior suggests that the gel–sol transition mechanism of the PVA–gelatin gel was not affected by the PVA concentration (Table 3). The gel–sol transition temperature stayed around
Table 4 Parameters of the straight line equation (y = A + Bx) and correlation coefficient (r2) evaluated by linear regression of the data of gel–sol (y) enthalpy in function of the PVA concentration in the blends. Samples/scan
A (kJ/g macromolecules)
B (102 kJ/g PVA)
r2
With glycerol, 1st scan With glycerol, 3rd scan Without glycerol, 1st scan Without glycerol, 3rd scan
13.2 13.0 14.1 13.4
0.17 0.16 0.19 0.19
0.986 0.996 0.830 0.917
26–27 °C, irrespective of the glycerol or PVA concentration, different to that observed by dynamic rheology (Table 1). Also, it was observed that the gel–sol transition enthalpy of the FFS (Fig. 5), calculated as the area under the peak of the Cp curve (Fig. 4), decreased linearly with the increase of the PVA concentration in the FFS; essentially independent of the presence of glycerol (Table 4). This linear relationship confirms that the effect of the PVA on the phase transitions (enthalpy and temperature) was due to substitution of gelatin by PVA, and so the concentration of the former was thus diluted in the samples. 3.4. Frequency sweep tests Overall, the measurement of G0 and G00 was confident only until 5 Hz. At 10 Hz, apparently, the behavior deviated from the visco-
Fig. 6. Storage (G0 ) and loss (G00 ) moduli as a function of frequency at 5 °C of FFS (gel) based on blends of gelatin and PVA (CevolÒ418), with (A and B) and without (C and D) glycerol and with different concentrations of PVA (indicated in the plots). Heating rate: 1 °C/min.
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elastic linear domain. Thus, examples of G0 and G00 behaviors as a function of frequency of the FFS determined at 5 °C, with and without glycerol, are shown in Fig. 6. It can be observed that, in all cases, the G0 values were more than one order of magnitude higher than those of G00 , typical behavior of viscoelastic gel-like solid materials (Ferry, 1980). This trend is probably related to the material structurisation due to network formation, which results in a more elastic system. Similar results have been observed in works on gels with 1%, 3% and 5% of gelatin (Zandi et al., 2007) and also with 15% of gelatin (Van den Bulcke et al., 2000). Overall, within the range of concentration values involved, G0 was not dependent on the frequency at low PVA concentrations in the blend, presenting a slight tendency to increase with the frequency at high PVA concentrations, mainly between 30% and 50% (Fig. 6). Systems that form ‘‘true gels” show G0 values practically independent of the frequency, as for the FFS based on pure gelatin (0% PVA), indicating that the deformation imposed in the network structure is entirely reversible. Then, the increase in the PVA concentration in the blend, decreased the gelatin gel strength, due to its dilution as previously described, resulting in further frequency dependence of G0 . The effect of the increase of the PVA concentration on the G0 values was more pronounced. Independently of the G0 behavior as a function of frequency, it was observed that overall, the values of G0 decreased from 200–300 to 2–6 Pa with the increase in the PVA concentration from 0% to 50% in the blend. From a molecular standpoint, it is well established that the elastic character is a function of the number of effective chains participating in the formation of a network structure (Gómez-Giullén et al., 2007). Thus, the decrease in G0 values as a function of the PVA concentration, was a consequence of the presence of a lower amount of interacting chains of gelatin due to a single dilution effect, or else, to the occurrence of macromolecular interactions involving the PVA and gelatin, since the hydroxyl groups in PVA, could interact by means of hydrogen bonds with some polar functional residues of the gelatin amino acids (Silva et al., 2008; Maria et al., 2008), and thus, avoiding the renaturation of this protein (Sobral and Habitante, 2001). Besides the gel-like character of the FFS, it is always interesting to analyze the results of G00 , where a strong effect of the PVA concentration in the blend was observed (Fig. 6). Overall, it was observed a trend of decreasing the G00 values at low frequencies, with an increase in its values as the frequency increased, giving place to a minimum. The position of this minimum was affected by the PVA, i.e., an increase in the PVA concentration caused a shift of the minimum to lower values of frequency, in such a way that the G00 curve of FFS with 50% of PVA, probably presented this minimum out of the scale studied (<0.1 Hz). According to Gilsenan and Ross-Murphy (2000), this behavior of G00 , with a minimum in function of the frequency, could be considered as typical of gelatin based systems, and may be associated with relaxation processes that occur at large time scales. On another hand, in pure PVA solutions, a monotonic increase of G00 in function of the frequency was observed (Choi et al., 2001). 4. Conclusions The effect of the PVA on the viscosity, phase transitions (gel–sol, sol–gel transitions) and viscoelastic properties of film forming solutions based on blends of gelatin and PVA, was due to protein dilution, i.e., by simple substitution, when a concentration of 2 g macromolecules/100 g of film forming solution was fixed. Furthermore, the glycerol did not affect any results obtained, probably because it was very diluted, about 0.5% in the FFS, provided the glycerol concentration was chosen in function of the
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macromolecules weight. On this dilution condition, the interactions among the macromolecules could occur especially with water molecules, rather than with those of glycerol. But, this is not a true problem for film properties, because during the film production, water used as a solvent will evaporate, impacting on the concentration of the plasticizer (respect to the macromolecules), and thus favoring the plasticizer–macromolecules interactions. On application of dynamic rheological tests, all FFS involved showed physical gel-like behavior, whose strength was affected by PVA. Acknowledgments To FAPESP for the research grants (05/57781-8) and the PV fellowship for JSF (05/54952-6), and to CNPq for PQI fellowship of PJAS. JSF acknowledges the Instituto Politécnico Nacional in Mexico. References Arvanitoyannis, I.S., 2002. Formation and properties of collagen and gelatin films and coatings. In: Gennadios, A. (Ed.), Protein-based Films and Coatings. CRC Press, Boca Raton, pp. 275–304. Barreto, P.L.M., Roeder, J., Crespo, J.S., Maciel, G.R., Terenzi, H., Pires, A.T.N., Soldi, V., 2003. Effect of concentration, temperature and plasticizer of sodium caseinate and sodium caseinate/sorbitol solutions and glass transition of their films. Food Chemistry 82 (3), 425–431. Carvalho, R.A., Moraes, I.C.F., Bergo, P.V.A., Kamimura, E.S., Habitante, A.M.Q.B., Sobral, P.J.A., 2009. Study of some physical properties of biodegradable films based on blends of gelatin and poly(vinyl alcohol) using a response-surface methodology. Materials Science and Engineering C 29, 485–491. Cheow, C.S., Norizah, M.S., Kyaw, Z.Y., Howell, N.K., 2007. Preparation and characterization of gelatins from the skins of sin croaker (Johnius dussumieri) and shortfin scad (Decapterus macrosoma). Food Chemistry 101, 386–391. Chiellini, E., Cinelli, P., Fernandes, E.G., Kenawy, E.S., Lazzeri, A., 2001a. Gelatinbased blends and composites. Morphological and thermal mechanical characterization. Biomacromolecules 2, 806–811. Chiellini, E., Cinelli, P., Imam, S.H., Mao, L., 2001b. Composite films based on biorelated agro-industrial waste and poly(vinyl alcohol) preparation and mechanical properties characterization. Biomacromolecules 2, 1029–1037. Chiellini, E., Cinelli, P., Corti, A., Kenawy, E.R., 2001c. Composite films based on waste gelatin: thermal–mechanical properties and biodegradation testing. Polymer Degradation and Stability 73, 549–555. Cho, S.M., Gu, Y.S., Kim, S.B., 2005. Extracting optimization and physical properties of yellowfin tuna (Thunnus albacares) skin gelatin compared to mammalian gelatins. Food Hydrocolloids 19, 221–229. Choi, J.H., Seok, W., Ko, S.W., 2001. Rheological properties of syndiotacticity-rich ultrahigh molecular weight poly(vinyl alcohol) dilute solution. Journal of Applied polymer Science 82, 569–576. Cuq, B., Aymard, C., Cuq, J.L., Guilbert, S., 1995. Edible packaging films based on fish myofibrillar proteins: formulation and functional properties. Journal of Food Science 60 (6), 1369–1374. Ferry, J.N., 1980. Viscoelastic Properties of Polymers. John Wiley and Sons Inc., New York. Fitzsimons, S.M., Mulvihill, D.M., Morris, E.R., 2008. Segregative interactions between gelatin and polymerized whey protein. Food Hydrocolloids 2, 485– 492. Gennadios, A., Mchugh, T.H., Weller, C.L., Krochta, J.M., 1994. Edible coating and films based on proteins. In: Krochta, J.M., Baldwin, E.A., Nisperos-Carriedo, M.O. (Eds.), Edible Coatings and to Improve Food Quality. Technomic, Lancaster, pp. 201–277. Gilsenan, P.M., Ross-Murphy, S.B., 2000. Viscoelaticity of thermoreversible gelatin gels from mammalian and piscine collagens. Journal of Rheology 44 (4), 871– 883. Gómez-Giullén, M.C., Ihl, M., Bifani, V., Silva, A., Montero, P., 2007. Edible films made from tuna-fish gelatin with antioxidant extracts of two different murta ecotypes leaves (Ugni molinae Turcz). Food Hydrocolloids 21, 1133–1143. Habitante, A.M.B.Q., Sobral, P.J.A., Carvalho, R.A., Feria, J.S., Bergo, P.V.A., 2008. Phase transitions of cassava starch dispersions prepared with glycerol solutions. Journal Thermal Analysis and Calorimetry 93, 599–604. Maria, T.M.C., Carvalho, R.A., Sobral, P.J.A., Habitante, A.M.B.Q., Solorza-Feria, J., 2008. The effect of the degree of hydrolysis of the PVA and the plasticizer concentration on the color, opacity, and thermal and mechanical properties of films based on PVA and gelatin blends. Journal of Food Engineering 87, 191– 199. Matsumura, S., Tomizawa, N., Toki, A., Nishikawa, K., Toshima, K., 1999. Novel poly(vinyl alcohol)-degrading enzyme and the degradation mechanism. Macromolecules 32 (23), 7753–7761. Mendieta-Taboada, O.W.M., Sobral, P.J.A., Carvalho, R.A., Habitante, A.M.B.Q., 2008. Thermomechanical properties of biodegradable films based on blends of gelatin and poly(vinyl alcohol). Food Hydrocolloids 22, 1485–1492.
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Menegalli, F.C., Sobral, P.J.A., Roques, M., Laurent, S., 1999. Characteristics of gelatin biofilms in relation to drying process conditions near melting. Drying Technology 17, 1697–1706. Peressini, D., Bravin, B., Lapasin, R., Toki, A., Nishikawa, K., Sensidoni, A., 2003. Starch-methylcellulose based edible films: rheological properties of filmforming dispersions. Journal of Food Engineering 59, 25–32. Sato, A.C.H., Oliveira, P.R., Cunha, R.L., 2008. Rheology of mixed pectin solutions. Food Biophysics 3 (1), 100–109. Silva, G.G.D., Sobral, P.J.A., Carvalho, R.A., Bergo, P.V.A., Mendieta-Taboada, O.W., Habitante, A.M.Q.B., 2008. Biodegradable films based on blends of gelatin and poly(vinyl alcohol): effect of PVA type or concentration on some physical properties of films. Journal of Polymers and the Environment 16, 276–285. Slade, L., Levine, H., 1987. Polymer-chemical properties of gelatin in foods. In: Pearson, A.M., Dutson, T.R., Bailey, A.J. (Eds.), Advances in Meat Research, Collagen as a Food. Elsevier Applied Science, London, pp. 251–266. Sobral, P.J.A., Habitante, A.M.Q.B., 2001. Phase transitions of pigskin gelatin. Food Hydrocolloids 15 (4–6), 377–382. Sobral, P.J.A., Menegalli, F.C., Hubinger, M.D., Roques, M.A., 2001. Mechanical, water vapor barrier and thermal properties of gelatin based edible films. Food Hydrocolloids 15 (4–6), 423–432.
Sudhamani, S.R., Prasad, M.S., Sankar, U.K., 2003. DSC and FTIR studies on gellan and polyvinyl alcohol (PVA) blends films. Food Hydrocolloids 17, 245–250. Tharanathan, R.N., 2003. Biodegradable films and composite coatings: past, present and future. Trends in Food Science and Technology 14, 71–78. Van den Bulcke, A.I., Bogdanov, B., Rooze, N.D., Schacht, E.H., Cornelissen, M., Berghmans, H., 2000. Structural and rheological properties of methacrylamide modified gelatin hydrogels. Biomacromolecules 1 (1), 31–38. Xiong, Y.L., Blanchard, S.P., 1994. Rheological properties of salt-soluble protein from white and red skeletal muscles. Journal of Agricultural and Food Chemistry 42 (8), 1624–1628. Yamaura, K., Naioth, M., 2002. Preparation of high performance films from poly(vinyl alcohol)/NaCl/H2O systems. Journal of Materials Science 37, 705– 708. Zandi, M., Mirzadeh, H., Mayer, C., 2007. Early stages of gelation in gelatine solution detected by dynamic oscillating rheology and nuclear magnetic spectroscopy. European Polymer Journal 43, 1480–1486.
Contents lists available at ScienceDirect
Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Film forming solutions based on gelatin and poly(vinyl alcohol) blends: Thermal and rheological characterizations Izabel C.F. Moraes a, Rosemary A. Carvalho a, Ana Mônica Q.B. Bittante a, Javier Solorza-Feria b, Paulo J.A. Sobral a,* a b
Department of Food Engineering, FZEA, University of São Paulo, 13635-900 Pirassununga/SP, Brazil Centro de Desarrollo de Productos Bióticos del IPN, Carr. Yautepec-Jojutla, Km 6, calle CEPROBI No. 8, Col. San Isidro, C.P. 62731 Yautepec, Morelos, Mexico
a r t i c l e
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Article history: Received 6 March 2009 Received in revised form 17 May 2009 Accepted 14 June 2009 Available online 21 June 2009 Keywords: Viscoelasticity Viscosity Gel Helix-coil transition Differential scanning microcalorimetry Glycerol
a b s t r a c t The objective of this work was to study the rheological and thermal properties of film forming solutions (FFS) based on blends of gelatin and poly(vinyl alcohol) (PVA). The effect of the PVA concentration and plasticizer presence on the flow behavior, and viscoelastic and thermal properties of FFS was studied by steady-shear flow and oscillatory experiments, and also, by microcalorimetry. The FFS presented Newtonian behavior at 30 °C, and the viscosity was not affected neither by the PVA concentration nor by the plasticizer. All FFS presented a phase transition during tests applying temperature scanning. It was verified that the PVA affected the viscoelastic properties of FFS by dilution of gelatin. This behavior was confirmed by microcalorimetric analysis. The behaviors of the storage (G0 ) and loss (G00 ) moduli as a function of frequency of FFS obtained at 5 °C were typical of physical gels; with the G0 higher than the G00 . The strength of the gels was affected by the PVA concentration. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Packages made with synthetic plastic films display overall excellent functional properties, but usually they are non-biodegradable, and therefore lead to environmental pollution, which pose serious ecological problems (Tharanathan, 2003). Thus, researches on development of biodegradable films have increased in the last years. After all, biodegradability is not only a functional requirement but also an important environmental attribute (Tharanathan, 2003). Several raw materials may be used to obtain biodegradable films (Tharanathan, 2003), being outstanding the biopolymers from renewable sources such as gelatin (Arvanitoyannis, 2002), and also synthetic polymers, as the poly(vinyl alcohol) (PVA) (Matsumura et al., 1999). More recently, biodegradable films have been developed using mixtures of gelatin and PVA (Chiellini et al., 2001a,b,c). Gelatin is a protein of animal origin, resulting from the acid or basic hydrolysis of collagen coming from bones, bovine and porcine skins, and from connective tissues (Gennadios et al., 1994; Arvanitoyannis, 2002). This macromolecule has been used in films production, most probably due to its outstanding filmogenic properties and its high production volumes at competitive prices (Sobral et al., * Corresponding author. Tel.: +55 19 35654192; fax: +55 19 35654114. E-mail address: [email protected] (P.J.A. Sobral). 0260-8774/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2009.06.023
2001). Gelatin has the capacity to form physical gels, i.e., thermoreversible gels. At a molecular level, gelatin gel formation involves a structural re-arrangement of the protein, implying the change from a disordered stage to a more ordered one, formed by triple helix structures (Slade and Levine, 1987; Arvanitoyannis, 2002). The poly(vinyl alcohol) (PVA) is produced from poly(vinyl acetate), by hydrolysis of the functional acetate groups (Yamaura and Naioth, 2002; Sudhamani et al., 2003). The PVA is a synthetic polymer, water soluble, non-toxic, which has been used in various materials in the medical and biomedical areas (Yamaura and Naioth, 2002). It also has excellent film forming properties (Sudhamani et al., 2003). Biodegradable films from gelatin and PVA have been developed by the wet method, that is to say, by casting and dehydration of a film forming solution (FFS) (Chiellini et al., 2001a,b,c; Maria et al., 2008; Mendieta-Taboada et al., 2008; Silva et al., 2008; Carvalho et al., 2009). According to Cuq et al. (1995) and Peressini et al. (2003), the knowledge of the FFS rheological properties by stationary and dynamic tests, is important for the design and processing of films by casting. It may be found in the specialized literature that diverse studies on the rheological behavior, including the viscoelasticity, have been carried out with relatively highly viscous FFS (Cuq et al., 1995; Barreto et al., 2003; Peressini et al., 2003). However, few studies have been carried out about the characterization of low viscosity FFS, produced usually with low concentration of macromolecules (Peressini et al., 2003; Gómez-Giullén et al.,
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2007). Within this context, the purpose of this work was to study the rheological and thermal properties of relatively diluted film forming solutions, based on blends of gelatin and PVA. The main interest was to verify the effect of the PVA concentration and plasticizer presence on the flow behavior, and viscoelastic and thermal properties of FFS.
2. Materials and methods
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2.5. Dynamic tests For the dynamic tests, the extent of the linear viscoelastic region was determined by performing a strain (0–20%) sweep in FFS both as a gel (5 °C) and as a sol (30 °C), in all cases at 1 Hz of frequency. Thus, according to the results of these tests, the strain value used for all following dynamic tests was fixed at 4%, within the linear viscoelastic domain. The viscoelastic parameters determined in these trials were the storage or elastic modulus (G0 ) and the loss or viscous modulus (G00 ).
2.1. Materials (a) Temperature sweep tests The macromolecules used for film forming solutions (FFS) production were: pigskin gelatin (bloom 242–248; molecular weigh 5.2 104 Da; moisture content = 9.3%) supplied by Gelita South America (São Paulo, Brazil), and poly(vinyl alcohol) with a degree of hydrolysis (DH) of 91.8%, molecular weight (Mn, furnished by supplier) of 85–124 kDa and viscosity of 17.6 cp (CevolÒ418) supplied by Celanese (Dallas, USA). Glycerol (Synth) was used as plasticizer. 2.2. Sample preparation The film forming solutions (FFS) were produced from a mixture of gelatin (solution A) and PVA (solution B) solutions. The solution A was prepared as follows: gelatin (different concentrations) was hydrated for 30 min, and then dissolved at 55 °C (Sobral et al., 2001) using a thermostatic bath (Marconi, Model TE 184). When necessary, glycerol was added and the solution was held at 55 °C for further 15 min. To prepare the solution B, the PVA (different concentrations) was first homogenized in distilled water and then dissolved at 95 °C (Chiellini et al., 2001a) with magnetic stirring (Tecnal-TE085). Solutions A and B were then mixed and homogenized for 15 min at room temperature with magnetic stirring, so as to produce FFS with 2 g macromolecules/100 g FFS with different proportions of PVA and gelatin (0, 10, 20, 30, 40 and 50 g PVA/100 g macromolecules, corresponding to 0:100; 10:90; 20:80; 30:70; 40:60 and 50:50 of PVA:gelatin, respectively), and plasticizer concentrations of 0 and 25 g glycerol/100 g macromolecules. More details on that preparation can be observed on previous works (Maria et al., 2008; Mendieta-Taboada et al., 2008). These FFS were immediately analyzed to determine its rheological behavior by steady-shear tests, and dynamic tests, with temperature and frequency sweeps. Also, the phase properties of FFS were determined by differential scanning microcalorimetry (microDSC). All tests in this study were performed at least in triplicate. 2.3. Rheological measurements Rheological tests were carried out in a rheometer (AR2000 Advanced Rheometer; TA Instruments, New Castle, DE, USA) using a cone and plate sensor geometry (cone angle 4°, 60 mm diameter). This equipment controlled the temperature with a peltier system. The results were analyzed using the software Rheology Advantage Data Analysis V.5.3.1 (TA Instruments). Before undertaking any rheological tests, all samples remained at rest for at least 5 min between the upper cone and plate geometry and the lower fixed plate, to allow the relaxation of stresses induced during sample loading (Peressini et al., 2003). To avoid water losses due to evaporation, the measurement system was coated with a solvent trap accessory.
Firstly, the FFS was heated up to 50 °C without a predetermined heating rate program, beginning the tests when that measurement temperature was attained. The essays were actually divided in two parts, a test was run while the sample was cooled down from 50 to 5 °C, remaining at this latter temperature for 5 min., and then another test was run while heating up the sample to 50 °C, always at a scan rate of 1 °C/min. From the results of these tests, the G0 and G00 values as a function of temperature were used to determine the following parameters: gelling (Tsol–gel) and melting (Tgel–sol) temperatures, calculated as the temperature where G0 changed drastically as an inflexion point; and the viscoelastic moduli (G0 and G00 ) of the gel, calculated at 5 °C (Gómez-Giullén et al., 2007). (b) Frequency sweep tests Some tests were carried out in the sol domain, but the results (G0 values) were very low, i.e., beyond the limit of sensitivity of the equipment. Thus, it was decided to run the frequency sweep tests at 5 °C, solely in the gel domain of samples. This temperature was attained by cooling down the FFS at 1 °C/min. Then, samples were subjected to sweep tests from 0.1 to 10 Hz (Van den Bulcke et al., 2000; Peressini et al., 2003). In these tests, the results were presented as G0 and G00 as a function of frequency. 2.6. Thermal analysis Thermal analyses were carried out using a differential scanning microcalorimeter (nanoDSC III, CSC 6300, Calorimetry Sciences Corporation, UT, USA). To use this technique, the FFS had to be diluted (101) in distilled water because of the equipment sensitivity (1 lcal.). This solution was then added into the measurement cell using a micropipette. Distilled water was added to the reference cell. These cells were pressurized at 3 atm to avoid water evaporation during heating. The raw data were analyzed using the software Cpconvert v.2.1.1, Cpcalc Analysis Tools (CSC – Calorimetry Sciences Corporation). Before starting these analyses, the cells were cooled down to 2 °C without any control of the cooling rate, remaining at that temperature (isothermal) for 10 min. Then, the cells were subjected to the following analyses program, always at 1 °C/min: heating up from 2 to 80 °C (scan 1), staying isothermally at 80 °C for 10 min, cooling down from 80 to 2 °C (scan 2), remaining at the latter temperature for 10 min, and then heating up again from 2 to 80 °C (scan 3). The phase transition temperature was estimated as the peak temperature of the endothermal phenomenon, and the phase transition enthalpy was calculated as the area under this same peak (Sobral and Habitante, 2001; Habitante et al., 2008).
2.4. Stationary state tests 2.7. Statistical analyses Steady-shear measurements were performed in an extended shear rate range (0–200 s1), at 30 °C. According to previous tests, at this temperature the FFS was always in the sol domain.
Statistical analyses were carried out using the SAS program (Version 9.1, SAS Institute Inc., Cary, NC, USA) and in case of signif-
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icance, the differences between means were determined by the Duncan multiple test (p < 0.05). 3. Results and discussion 3.1. Flow behavior All results of the steady state tests showed similar behavior; the relationship between the shear stress and shear rate was linear. Examples of these profiles are shown in Fig. 1. The average viscosity value of FFS was about 7.1 ± 0.1 mPa s, irrespective of the concentrations of the PVA or glycerol. This Newtonian behavior appeared certainly because the FFS were in fact diluted solutions (2%). In these cases, the behavior is mainly linked to the macromolecules–water interactions rather than to the macromolecules–macromolecules ones (Barreto et al., 2003). The organization of the polypeptides (gelatin) in the already known triple helix, as well as the associations of these organized sequences, could take place, depending on factors such as gelatin chemical structure, concentration and temperature, affecting the FFS rheological behavior. This last tendency of macromolecules interaction has been observed in more concentrated systems, with a consequent non-Newtonian behavior (Xiong and Blanchard, 1994; Cuq et al., 1995; Peressini et al., 2003; Sato et al., 2008). Fitzsimons et al. (2008) determined flow curves for both unpolymerized and polymerized whey protein isolate (6% WPI, pH 7), alone and mixed with 1% and 2% of type B gelatin and observed Newtonian behavior, with viscosity values around 2 mPa s. When these authors mixed the WPI with 3% or 4% of gelatin, some slight shear-thinning behavior was noticed. Barreto et al. (2003) studied sodium caseinate FFS as a function of biopolymer concentration, temperature and plasticizer content and also observed Newtonian behavior; consistent with this work, with no effect of the plasticizer (sorbitol) concentration on the viscosity of diluted FFS. The leveling of irregularities in liquid coating (or in FFS casting), depends mainly, on their rheological properties (Peressini et al., 2003). According to Cuq et al. (1995), to cast a high viscosity FFS is very difficulty and needs a dispersing machine (spreader). Nevertheless, the FFS studied in this work had low viscosity values, which readily allowed casting these solutions by free flow (Maria et al., 2008; Mendieta-Taboada et al., 2008; Silva et al., 2008; Carvalho et al., 2009).
Fig. 1. Examples of flow curves determined at 30 °C, of film forming solutions with 0% of PVA with () and without glycerol (j), and with 50% of PVA without glycerol (h).
3.2. Scanning temperature in dynamic rheological tests Similar rheological profiles were observed in all temperature sweep results (Figs. 2 and 3); within the range studied, both moduli (G0 and G00 ) had higher values at low than at relatively high temperatures. Between these two domains, an inflexion was observed in both moduli. These behaviors are typical of physical gels presenting a sol–gel (cooling, Tsol–gel) and/or a gel–sol (heating, Tgel– sol) transition, such as gelatin. Moreover, as expected, the viscous modulus (G00 ) was higher than the elastic modulus (G0 ) over the sol domain temperature and when the systems showed a gel character, it was also observed that the stiffness (elastic character) increased with gelatin concentration in the blend (Figs. 2 and 3). Similar profiles have been observed by several authors in gelatin based systems (Van den Bulcke et al, 2000; Cho et al., 2005; Gómez-Giullén et al., 2007). Overall, a sol–gel or a gel–sol transition of proteins is a multistep thermodynamic process that involves a three-dimensional network structure formation (Xiong and Blanchard, 1994). That is to say, the gelation of gelatin is the result of intermolecular interactions among protein molecules usually involving hydrogenbonding, electrostatic and hydrophobic interactions (Gennadios et al., 1994). Gelatin is then, an entangled polymer network that, depending upon the specific temperature and time of application, is interspersed with certain number of crosslinks that affects its rheological, and mechanical properties. The temperatures of the sol–gel and gel–sol transitions, fitted as the temperature where the peak of the curve of the first derivative of G0 occurred, were affected by the PVA concentration, without a notable effect due to glycerol (Table 1). Peressini et al. (2003), working with methylcellulose (MC) and glycerol solutions, also observed that the MC concentration had an effect much more significant on the viscoelastic properties of these solutions, than the glycerol concentration. Overall, the increase in the PVA concentration in the blend, caused an intense reduction (47%) in the Tsol–gel, which decreased from around 15 °C to about 8 °C, whereas the Tgel–sol decreased (14%) from around 29 °C to about 25 °C, irrespective of the presence of glycerol in the FFS. In consequence, an increase in the hysteresis was observed, this meant that as the PVA concentration in the blend increased, the difference between Tgel–sol and Tsol–gel also increased (Table 1). These behaviors may well be associated with the processes involved in the coil-helix/helix-coil transition, i.e., the gelling of the solution and the melting of the gel. The gelatin triple helix formation (coil-helix transition) is a slow process, which continues almost indefinitely, while the dissociation (helix-coil transition) of the triple helix is an equilibrium process linked with the transition temperature (Fitzsimons et al., 2008). Cho et al. (2005) have performed dynamic rheological studies, by running temperature sweeps in dispersions with 6.67% of gelatin from pig, and determined higher Tgel–sol (36.5 °C) and Tsol–gel (25.6 °C) values than those found in this work with gelatin without PVA (Table 1). Even though these authors have used a different heating rate (0.5 °C/min), their results could possibly be because they have worked with more concentrated systems. However, it has to be noted that the hysteresis (11 °C) found by Cho et al. (2005) was similar to the one observed in this work (13 °C). Moreover, Cheow et al. (2007), who worked with 6.67% bovine gelatin solutions, determined intermediate values of Tsol–gel (19.6 °C) and Tgel–sol (28.8 °C). The effect of the PVA concentration on the transition temperature may be explained from the results of the viscoelasticity rheological tests with pure PVA solutions (Fig. 2). It was seen that this polymer practically did not show elastic behavior (G0 ? 0) and also, it did not display any phase transition within the range of temperatures studied. This suggests that probably the effect of
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Fig. 2. Storage (G0 ) and loss (G00 ) moduli of FFS based on blends of gelatin and PVA (CevolÒ418) without glycerol, as a function of temperature. (A and C) cooling; (B and D) heating, both at 1 °C/min.
Fig. 3. Storage (G0 ) and loss (G00 ) moduli of FFS based on blends of gelatin and PVA (CevolÒ418) with glycerol, as a function of temperature. (A and C) cooling; (B and D) heating, both at 1 °C/min.
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Table 1 Transition temperatures Tsol–gel; Tgel–sol and DT (hysteresis) of film forming solutions based on gelatin and PVA (CevolÒ418) with 0 and 25 g of glycerol/100 g of macromolecules.a Concentration of PVA (g/100 g)b
0 10 20 30 40 50 a b
With glycerol
Without glycerol
Tsol–gel (°C)
Tgel–sol (°C)
DT (°C)
Tsol–gel (°C)
Tgel–sol (°C)
DT (°C)
15.1 ± 0.3a 15.1 ± 0.4a 13.8 ± 0.3b 12.7 ± 1.0c 10.6 ± 0.5d 8.4 ± 0.5e
28.5 ± 0.3a 28.3 ± 0.1a 27.8 ± 0.2b 27.1 ± 0.4c 26.6 ± 0.6d 24.7 ± 0.1e
13.4 ± 0.2c 13.2 ± 0.4c 14.1 ± 0.4b 14.4 ± 0.9b 16.0 ± 0.6a 16.3 ± 0.4a
15.3 ± 0.2a 14.4 ± 0.2b 13.8 ± 0.4c 12.7 ± 0.1d 10.8 ± 0.2e 8.5 ± 0.4f
28.6 ± 0.3a 28.1 ± 0.3b 27.8 ± 0.3b.c 27.5 ± 0.2c 26.4 ± 0.4d 25.3 ± 0.4e
13.3 ± 0.2e 13.7 ± 0.2d 14.0 ± 0.4d 14.8 ± 0.2c 15.6 ± 0.4b 16.8 ± 0.3a
Different letters in the same column indicate significant differences (p < 0.05) among the average values, by application of the Duncan test using the SAS program. g PVA/100 g macromolecules.
the PVA on gelatin gelation, could have been nothing else but a consequence of the gelatin dilution in the blend, and thus, interfering with the continuity of the gel that this protein forms, by obstructing the conformational transition of gelatin molecules from random coils, to triple helical structures which joins several chains. Previous authors found that the gelatin gelation temperature was highly dependent on this protein concentration in solution, and in the present case, therefore, on the PVA concentration in the blend (Gómez-Giullén et al., 2007). With the reduction of the gelatin concentration in solution, the intermolecular interactions during the ‘‘helix” structure formation became less probable (Van den Bulcke et al., 2000). However, as previously mentioned, the occurrence of certain type of interaction between the PVA and gelatin, which could in consequence avoid gelatin junctions formation and therefore, that of the gelatin network cannot be disregarded. Van den Bulcke et al. (2000) studied the gelation of gelatin reticulated with meta-acrylamide, with different degrees of reticulation, and obtained results apparently similar to those presented in Figs. 2 and 3. The increase of the reticulation degree caused a decrease of the Tgel–sol, similar trend to that observed in this work (Table 1). Furthermore, these authors verified that the reticulation degree of the gelatins also affected the gels rigidity (stiffness), i.e., the values of the storage modulus (G0 ) of the gels at temperatures lower than the Tgel–sol, decreased as the gelatin reticulation degree increased. Similar results respect to the effect of the PVA concentration in the blend were obtained in this work: in all cases studied, an increase in the PVA concentration caused a decrease in the G0 values determined at 5 °C (Table 2). According to GómezGiullén et al. (2007), the rigidity of the gelatin gel is also highly dependent of this protein concentration in solution. Thus, it may be considered that, with the increase of the PVA concentration in the FFS, there were less intermolecular associations between proteins, and consequently, a weaker gel was formed. The presence of the plasticizer (glycerol) in the FFS did not affect neither the transition temperatures (Table 1) nor the G0 values determined at 5 °C (Table 2). The glycerol is a low molecular weight molecule, that when added to polymeric materials in relatively high concentrations (e.g., 10–40 g/100 g polymer), modifies
the tridimensional organization of the polymeric matrix, decreasing the intermolecular attraction forces and consequently, increasing the free volume and the mobility of the polymeric chain. This is done via hydrogen bonds between its hydroxyl groups and the polar functional groups of some amino acid residues (Gennadios et al., 1994; Cuq et al., 1995; Sobral et al., 2001). However, in the FFS production of the present work, the plasticizer concentration was very low; about 0.5% (g glycerol/100 g of FFS). Thus, the polar functional groups of the gelatin amino acids should have interacted firstly with water molecules, which were in greater numbers than those of glycerol. The production of films by casting implies, initially, the FFS manufacture, where the biopolymer is found in the form of sol. After the application of the FFS on a support material and then subjecting the FFS to drying, film formation takes place. In an intermediate stage, especially in the case of films made from gelatin, the film will necessarily pass by a gel formation, with a semi-rigid tridimensional matrix constitution, which will involve and immobilize the plasticizer and the solvent (water). Then, with the water evaporation by dehydration, the plasticizer will become more concentrated. Thus, the formation of the biopolymeric matrix will take place via electrostatic, hydrophobic, and Van der Walls interactions, as well as hydrogen bonds among adjacent biopolymer chains and between these and the plasticizer (Cuq et al., 1995). Effectively, the transition (Tsol–gel 6 15.3 °C; Tgel–sol 6 28.6 °C) temperatures of the studied FFS (Table 1) were below the usual drying temperature (30 °C) (Maria et al., 2008; Mendieta-Taboada et al., 2008). Thus, in that condition, it might be considered that the FFS could not be a real gel. But, in fact, the gel formation could have occurred during the application/drying of the FFS, either because the air wet bulb temperature was lower than the respective dry bulb temperature; or because a material concentration occurred during the drying process, which implied the increase of the transition temperatures, as observed by Menegalli et al. (1999). 3.3. Scanning temperature in microcalorimetry To supplement the temperature sweep dynamic rheological tests, microcalorimetric analyses were undertaken to the same
Table 2 Storage modulus (G0 ) of gels based on gelatin and PVA (2 g of macromolecules/100 g of FFS), with 0 and 25 g of glycerol/100 g of macromolecules, at 5 °C.a Concentration of PVA (g/100 g)b
0 10 20 30 40 50 a b
With glycerol
Without glycerol
Cooling (Pa)
Heating (Pa)
Cooling (Pa)
Heating (Pa)
198.1 ± 12.8a 131.6 ± 1.5b 84.8 ± 17.9c 19.4 ± 3.5d 12.0 ± 1.2d 2.2 ± 0.4e
296.5 ± 5.2a 216.4 ± 1.8b 145.1 ± 23.6c 42.5 ± 6.8d 24.8 ± 3.5e 8.0 ± 1.0f
206.0 ± 13.6a 132.7 ± 4.3b 81.8 ± 6.2c 38.5 ± 3.3d 12.8 ± 0.4e 2.3 ± 0.2f
305.9 ± 5.9a 212.2 ± 12.4b 136.9 ± 7.6c 71.0 ± 8.4d 27.8 ± 2.0e 7.7 ± 1.0f
Different letters in the same column indicate significant differences (p < 0.05) among the average values, by application of the Duncan test using the SAS program. g PVA/100 g macromolecules.
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previously described samples, diluted ten times due to equipment sensibility. In these analyses, the transition temperature was observed as a peak in the specific heat (Cp) variation as a function
of temperature (Fig. 4), in the heating thermograms. These endothermal peaks could be assigned to the gelatin triple helix dissociation, the so-called gel melting. It is interesting to note that the Cp
Fig. 4. Specific heat of diluted film forming solutions based on blends of gelatin and PVA, with (A and B) and without (C and D) glycerol and with different concentrations of PVA (indicated in the plotts): B and D – 1st scan; A and C – 3rd scan, obtained by microcalorimetry. Heating rate: 1 °C/min.
Table 3 Transition temperature (Tgel–sol) of film forming solutions based on gelatin and PVA (CevolÒ418) determined by microcalorimetry.a Concentration of PVA (g/100 g)b
With glycerol Tgel–sol (°C) (1° scan)
Tgel–sol (°C) (3° scan)
Tgel–sol (°C) (1° scan)
Tgel–sol (°C) (3° scan)
0 10 20 30 40 50
26.8 ± 0.5a 26.6 ± 0.5a 28.4 ± 3.0a 26.8 ± 0.4a 28.2 ± 2.7a 26.9 ± 0.3a
26.4 ± 0.1a 26.2 ± 0.2a 26.4 ± 0.0a 26.3 ± 0.1a 26.3 ± 0.1a 26.4 ± 0.3a
26.9 ± 0.4a 27.8 ± 2.5a 27.0 ± 0.1a 28.6 ± 2.9a 27.1 ± 0.5a 30.1 ± 2.7a
26.7 ± 0.2a,b 26.7 ± 0.3a,b 26.9 ± 0.1a 26.4 ± 0.4b 26.7 ± 0.1a,b 26.8 ± 0.2a
a b
Without glycerol
Different letters in the same column indicate significant differences (p < 0.05) among the average values, by application of the Duncan test using the SAS program. g PVA/100 g macromolecules.
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Fig. 5. Enthalpy (DH) of gel–sol transition as a function of PVA concentration (CPVA) in the blend of the film forming solutions of gelatin and PVA. (A) 1st scan; (B) 3rd scan.
peaks were almost independent of the PVA concentrations in the blends. This behavior suggests that the gel–sol transition mechanism of the PVA–gelatin gel was not affected by the PVA concentration (Table 3). The gel–sol transition temperature stayed around
Table 4 Parameters of the straight line equation (y = A + Bx) and correlation coefficient (r2) evaluated by linear regression of the data of gel–sol (y) enthalpy in function of the PVA concentration in the blends. Samples/scan
A (kJ/g macromolecules)
B (102 kJ/g PVA)
r2
With glycerol, 1st scan With glycerol, 3rd scan Without glycerol, 1st scan Without glycerol, 3rd scan
13.2 13.0 14.1 13.4
0.17 0.16 0.19 0.19
0.986 0.996 0.830 0.917
26–27 °C, irrespective of the glycerol or PVA concentration, different to that observed by dynamic rheology (Table 1). Also, it was observed that the gel–sol transition enthalpy of the FFS (Fig. 5), calculated as the area under the peak of the Cp curve (Fig. 4), decreased linearly with the increase of the PVA concentration in the FFS; essentially independent of the presence of glycerol (Table 4). This linear relationship confirms that the effect of the PVA on the phase transitions (enthalpy and temperature) was due to substitution of gelatin by PVA, and so the concentration of the former was thus diluted in the samples. 3.4. Frequency sweep tests Overall, the measurement of G0 and G00 was confident only until 5 Hz. At 10 Hz, apparently, the behavior deviated from the visco-
Fig. 6. Storage (G0 ) and loss (G00 ) moduli as a function of frequency at 5 °C of FFS (gel) based on blends of gelatin and PVA (CevolÒ418), with (A and B) and without (C and D) glycerol and with different concentrations of PVA (indicated in the plots). Heating rate: 1 °C/min.
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elastic linear domain. Thus, examples of G0 and G00 behaviors as a function of frequency of the FFS determined at 5 °C, with and without glycerol, are shown in Fig. 6. It can be observed that, in all cases, the G0 values were more than one order of magnitude higher than those of G00 , typical behavior of viscoelastic gel-like solid materials (Ferry, 1980). This trend is probably related to the material structurisation due to network formation, which results in a more elastic system. Similar results have been observed in works on gels with 1%, 3% and 5% of gelatin (Zandi et al., 2007) and also with 15% of gelatin (Van den Bulcke et al., 2000). Overall, within the range of concentration values involved, G0 was not dependent on the frequency at low PVA concentrations in the blend, presenting a slight tendency to increase with the frequency at high PVA concentrations, mainly between 30% and 50% (Fig. 6). Systems that form ‘‘true gels” show G0 values practically independent of the frequency, as for the FFS based on pure gelatin (0% PVA), indicating that the deformation imposed in the network structure is entirely reversible. Then, the increase in the PVA concentration in the blend, decreased the gelatin gel strength, due to its dilution as previously described, resulting in further frequency dependence of G0 . The effect of the increase of the PVA concentration on the G0 values was more pronounced. Independently of the G0 behavior as a function of frequency, it was observed that overall, the values of G0 decreased from 200–300 to 2–6 Pa with the increase in the PVA concentration from 0% to 50% in the blend. From a molecular standpoint, it is well established that the elastic character is a function of the number of effective chains participating in the formation of a network structure (Gómez-Giullén et al., 2007). Thus, the decrease in G0 values as a function of the PVA concentration, was a consequence of the presence of a lower amount of interacting chains of gelatin due to a single dilution effect, or else, to the occurrence of macromolecular interactions involving the PVA and gelatin, since the hydroxyl groups in PVA, could interact by means of hydrogen bonds with some polar functional residues of the gelatin amino acids (Silva et al., 2008; Maria et al., 2008), and thus, avoiding the renaturation of this protein (Sobral and Habitante, 2001). Besides the gel-like character of the FFS, it is always interesting to analyze the results of G00 , where a strong effect of the PVA concentration in the blend was observed (Fig. 6). Overall, it was observed a trend of decreasing the G00 values at low frequencies, with an increase in its values as the frequency increased, giving place to a minimum. The position of this minimum was affected by the PVA, i.e., an increase in the PVA concentration caused a shift of the minimum to lower values of frequency, in such a way that the G00 curve of FFS with 50% of PVA, probably presented this minimum out of the scale studied (<0.1 Hz). According to Gilsenan and Ross-Murphy (2000), this behavior of G00 , with a minimum in function of the frequency, could be considered as typical of gelatin based systems, and may be associated with relaxation processes that occur at large time scales. On another hand, in pure PVA solutions, a monotonic increase of G00 in function of the frequency was observed (Choi et al., 2001). 4. Conclusions The effect of the PVA on the viscosity, phase transitions (gel–sol, sol–gel transitions) and viscoelastic properties of film forming solutions based on blends of gelatin and PVA, was due to protein dilution, i.e., by simple substitution, when a concentration of 2 g macromolecules/100 g of film forming solution was fixed. Furthermore, the glycerol did not affect any results obtained, probably because it was very diluted, about 0.5% in the FFS, provided the glycerol concentration was chosen in function of the
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