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Egg white-based bioplastics developed by thermomechanical processing
Journal of Food Engineering 82 (2007) 608–617 www.elsevier.com/locate/jfoodeng
Egg white-based bioplastics developed by thermomechanical processing A. Jerez a, P. Partal a,*, I. Martı´nez a, C. Gallegos a, A. Guerrero b a
Departamento de Ingenierı´a Quı´mica, Facultad de Ciencias Experimentales, Campus del Carmen, Universidad de Huelva, 21071 Huelva, Spain b Departamento de Ingenierı´a Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, 41012 Sevilla, Spain Received 14 September 2006; received in revised form 18 January 2007; accepted 11 March 2007 Available online 20 March 2007
Abstract The development of new protein-based biomaterials has been receiving increasing interest in the last few years. This paper is focused on the development of new bioplastic materials, based on wheat gluten and egg white proteins, manufactured by direct mixing of proteins with a plasticizer, glycerol, and, eventually, a thermal and moulding process which shapes the material and gives them suitable mechanical properties to be used as substitutive materials of synthetic polymers for definite applications. These rheological properties have been evaluated by shear rheology and dynamic mechanical thermal analysis on both intermediate process materials and final bioplastics. Moreover, the microstructure of the samples has been characterized by atomic force microscopy and modulated differential scanning calorimetry. Glycerol–protein mixtures have been proved to hold suitable rhelogical behaviour and thermosetting potential for further processing, by means of a compression-moulding process, to obtain bioplastics. Glycerol/egg white blends (0.5 ratio) can be more easily processed, because of their rheological behaviour at ca. 50 °C. On the contrary, glycerol/wheat gluten blends (0.5 ratio) need longer mixing times and more severe thermosetting conditions, although lower values of the bioplastic storage modulus are obtained in the whole range of temperature studied. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Egg white; Wheat gluten; Bioplastics; Rheology; Viscoelasticity; Processing
1. Introduction Biopolymers from agricultural sources are becoming a suitable alternative not only as biodegradable films, suitable for food packaging, but also as plastic stuffs which require improved mechanical properties. Proteins, lipids and polysaccharides have been used as biopolymer sources for many years. It is well-known that a wide variety of proteins are produced at huge scale for instance, wheat gluten, soy and pea proteins from vegetable resources; egg proteins, fish myofibrillars and wool keratin proteins from animal resources (Cuq, Gontard, & Guilbert, 1999; De Graaf, 2000; Domenek, Feuilloley, Gratraud, Morel, & Guilbert, 2004; Irissin-Mangata, Ge´rard, Bernard, & Gontard, 2001; Mine, 1995; Yamauchi & Yamauchi, 1997). In addi*
Corresponding author. E-mail address: [email protected] (P. Partal).
0260-8774/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2007.03.020
tion, biopolymers have the advantage of biodegradability, which adds an extra value to materials developed from them. Thus, protein-based biomaterials degradation rates have been proved to be among the rates of fast-degrading polymers, completely degrading in 50 days when buried in farmland soils (Domenek et al., 2004). Proteins used for food applications should be in their native form (non-denaturated state) in order to keep their functional properties. However, for non-edible applications, protein denaturation becomes an additional variable for the design of new materials, which properties may change depending on the denaturation degree of the proteins that conform them (De Graaf, 2000). Hen egg white proteins (albumen) have been often used as ingredients in food processing for their unique functional properties, such as gelling, foaming, heat setting and binding adhesion. Up to now, the structural and functional properties of egg white proteins have been studied
A. Jerez et al. / Journal of Food Engineering 82 (2007) 608–617
mainly focusing on the contribution of the major egg white protein, the ovalbumin. Ovalbumin constitutes over half of the egg white albumen. It is a monomeric phosphoglycoprotein and the only protein containing free sulfhydril groups, four, which are buried in the protein core. Heatinduced denaturation of ovalbumin results in the external exposure of these sulfhydril groups, accompanied by a decrease in the total sulfhydril content, due to the oxidation of SH groups to disulfide bonds (Van der Placken, Van Loey, & Hendrickx, 2005). A plasticizer is a film component, required to overcome film brittleness and helping to avoid chipping and cracking of films during subsequent handling and storage. Plasticizers are molecules with low molecular weight and low volatility, which modify the three-dimensional structure of proteins (Matveev, Grinberg, & Tolstoguzov, 2000). These plasticizers have a general behaviour that follows the Couchmann–Karasz relation using a heat capacity (DCp) value of 0.4 J/g K for wheat gluten (Pouplin, Redl, & Gontard, 1999). Comparing the plasticizer efficiency on a weight basis, a similar plastification has been obtained with plasticizers of different molecular structure (hydroxyl or amino groups). The plasticizing efficiency has been reported to be generally proportional to the molecular weight and inversely proportional to the percent of hydrophilic groups of the plasticizer (Welti-Chanes, BarbosaCa´novas, & Aguilera, 2002). Processing of films, coatings or other materials based on polymers derived from proteins requires three main steps: breaking of intermolecular bonds (non-covalent and covalent, if necessary) that stabilize the polymers in their native forms, by using chemical or physical rupturing agents; arranging and orienting mobile polymer chains into the desired shape; and, finally, allowing the formation of new intermolecular bonds and interactions to stabilize the three-dimensional network. The shape obtained in the second step is mainly maintained by eliminating the agents used to break the intermolecular bonds in the first step. Active sites for bond formation become free and close enough to each other to create new interactions, hydrogen bonds, hydrophobic interactions and disulfide bonds, forming a new three-dimensional network (Gennadios, 2002). The casting method, or physico-chemical method, for film processing is based on the above mentioned three steps, using a chemical reactant to disrupt disulfide bonds, dispersing, solubilizing proteins and finally drying it. Other authors, who have studied film formation by the casting method, have processed 50 lm thick samples, obtaining good quality films which could be used for food packaging in view of the properties they possess such as opacity, water vapour, oxygen and carbon dioxide permeability, and mechanical properties. However, the use of these films for other applications where they could substitute synthetic polymers is quite limited (Cherian, Gennadios, Weller, & Chinchoti, 1995; Herald, Gnanasambandam, McGuire, & Hachmeister, 1995).
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Another way of processing biomaterials is the mechanical method, or thermoplastic processing, which consists of mixing proteins and plasticizer to obtain a dough-like material. Therefore, the extrusion of proteins is, in general, only possible in a limited window of operating conditions and the material properties of the extrudates depend on the processing conditions in a complex way (Mitchell, Areas, Rasul, Coloma, & Della Valle, 1994; Redl, Morel, Bonicel, Vergnes, & Guilbert, 1999; Redl, Guilbert, & Morel, 2003). At this moment, most of the procedures for manufacturing egg white proteins films are based on the casting method (Gennadios, Weller, Hanna, & Froning, 1996). On the contrary, the use of a thermo-mechanical method to obtain an egg white-based bioplastic has not been previously investigated. This new method makes the bioplastic processing easier and may have an interesting industrial potential. As a result, the overall objective of this work was to obtain bioplastics from egg white proteins by means of a combined compression moulding/thermosetting procedure. This paper focuses on the effects that processing and a further thermal treatment exert on the thermo-mechanical properties and microstructure of the biomaterials obtained, and how they can be modified if the denaturation degree of the protein changes. The results obtained have been compared with those obtained from wheat gluten based bioplastics. 2. Materials and methods Wheat gluten was provided by RIBA S.A. Spray-dried egg white albumen was provided by OVOSEC S.A. Some compositional characteristics of these protein concentrates used as raw materials in this work are shown in Table 1. Glycerol, from Panreac Quı´mica, S.A. (Spain), was used as protein plasticizer. The thermo-mechanical processing was carried out in a torque-rheometer (Polylab, Haake, Germany) for the simulation of industrial processes in the lab or pilot plants, by means of the use of different modular tools. The mixer tool (Rheomix 3000p) enables the batch testing of many highly viscous substances and consists of a batch mixer fitted with two counter-rotating rollers, turning with different angular velocities (ratio 3:2). A detailed description of a common torque-rheometer can be found elsewhere (Dealy, 1982). Protein and glycerol were directly added in the mixing chamber before starting the mixing tests. Both torque Table 1 Some compositional characteristics of the protein concentrates used as raw materials in this work
Protein content (%) Lipids (%) Ashes (%) Moisture (%) Reducing sugars (%) pH (10% solution)
Wheat gluten (WG)
Egg white (EW)
83 1.5–2 0.7–0.8 8 – 6.88
73 – 6 8 0.1 7.10
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and temperature have been recorded during the mixing process. The mixing chamber can be considered adiabatic, as it was not cooled. The volume of the chamber (310 cm3) was filled with approximately 245 g of sample, corresponding to 80% filling ratio. The plasticizer concentration was 0.5 g glycerol/g protein for both gluten and albumen. The mixing process was always carried out at 50 rpm. Compression-moulded biomaterials were prepared by compressing the dough-like materials obtained after the mixing process at different gauge pressures, in a 50 10 3 mm mould. The equipment used to carry out this process was developed by our own laboratory facilities, consisting in a two electrical heated plates, with electronic temperature control, and a hydraulic pressure system. The compression-moulding process was carried out at three different temperatures 90, 120, and 140 °C, for wheat gluten bioplastics, and 60, 90 and 120 °C, for egg albumen bioplastics, during 10 min. Theses temperatures were above the maximum temperature reached during the mixing process and below the temperature at which the degradation of the blends takes place. Frequency sweep tests, from 0.01 to 100 rad/s, in oscillatory shear, at a constant stress within the linear viscoelastic region and a temperature of 25 °C, were conducted in a controlled stress rheometer Rheoscope (Haake, Germany), using a serrated parallel plate geometry, 20 mm diameter and 1–1.2 mm gap. Previously, stress sweep tests, at 6.28 rad/s, were carried out in order to identify the linear viscoelastic region of these materials. Temperature sweep tests, from 25 to 170 °C, were conducted in a controlled strain rheometer ARES (Rheometric Scientific, USA), using a serrated parallel plate geometry, 25 mm diameter and 1–1.2 mm gap. Measurements were performed at constant frequency (6.28 rad/s) and strain (1%, which was always within the linear viscoelastic region), selecting a 2 °C/min. temperature ramp. DMTA experiments were done with a Seiko DMS 6100 (Seiko Instruments, Japan), using 50 10 3 mm samples in double cantilever bending mode, according to the ASTM standard method D5023-01 (ASTM, 2001). All the experiments were carried out at constant frequency (1 Hz) and strain (within the linear viscoelastic region). The selected temperature ramp was 2 °C/min. MDSC experiments were performed with a Q100 (TA Instruments, USA), using 10–20 mg samples, in hermetic aluminium pans. An oscillation period of 60 s, with an amplitude of ±0.5 °C, and a heating rate of 5 °C/min, were selected. The sample was purged with a nitrogen flow of 50 ml/min. All the rheological and calorimetrical measurements were replicated at least three times. The microstructural characterization of the samples was carried out by means of atomic force microscopy (AFM) with a multimode AFM (Digital Instruments, Veeco Metrology Group; Inc., Santa Barbara, CA) connected to a Nanoscope_IV scanning probe microscope controller (Digital Instruments, Veeco Metrology Group Inc., Santa
Barbara, CA). All images were acquired in tapping mode using Veeco NanoprobeTM tips (Veeco Metrology Group Inc., Santa Barbara, CA). Atomic force microscopy (AFM) has arisen as one of the most powerful tools for determining the surface topography of biomacromolecules at nanometer or even subnanometer resolution (Engel, Lyubchenko, & Mu¨ller, 1999; Hansma et al., 1997; Silva, 2002). Furthermore, AFM images of biomaterials can give not only structural information but also knowledge on some materials properties of the sample. In some cases, new imaging modes generate AFM images that reflect material properties of the sample surface. An excellent example is the phase mode imaging, being a powerful extension of tapping mode AFM, which provides nanometer-scale information about surface structure and properties often not revealed by other scanning probe microscopy techniques. Phase imaging, which is simultaneously monitored with topography data, measures the phase shift between the oscillation driving the tip and the oscillation produced by the tip as it interacts with features on the sample surface. The phase lag is very sensitive to many material properties, such as variations in composition, adhesion, friction and viscoelasticity (Rosa-Zeiser, Weilandt, Hild, & Marti, 1997; Sheiko & Mo¨ller, 2001; Wahl, Stepnowski, & Unertl, 1998), and may show patterns of stiffness and/or hydrophobicity on the sample surface (Boussu et al., 2005; Magonov, Elings, & Whangbo, 1997). However, the interpretation of phase images is still at an early stage, so that there is currently no simple correlation between phase contrast and a single material property. 3. Results and discussion 3.1. Mechanical processing Processing of bioplastics implies the intimate mixing of plasticizers and proteins. Fig. 1 shows the evolution of both torque and temperature during this mixing process for glycerol/gluten (Fig. 1a) and glycerol/egg albumen (Fig. 1b) blends (plasticizer/protein ratio: 0.5). Three different regions may be observed (Fig. 1a). The first region, where no significant increase in torque is observed, corresponds to an induction period that takes place at low mixing times. In this first stage of processing, a dough-like material is obtained, which may be suitable for further thermo-mechanical treatments. In the second region, torque undergoes an exponential increase with mixing time up to a maximum value. Finally, an apparent torque decay is observed in the last region. Previous results obtained by the authors showed an important dependence of this torque profile on mixing conditions (Jerez, Partal, Martı´nez, Gallegos, & Guerrero, 2005). Two samples at different stages of the mixing process were selected in order to investigate the effect of the thermo-mechanical processing. The first one, denoted as sample G/WG-I, was taken out at the end of the first stage (after 10 min. mixing), whilst sample G/WG-II, which corresponds to the torque decay stage, was taken after 45 min. of processing. For each one of
A. Jerez et al. / Journal of Food Engineering 82 (2007) 608–617
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Fig. 1. Evolution of torque (a and b) and temperature (c) during the mixing process of proteins and plasticizers (plasticizer/protein ratio: 0.5). (a) glycerol and wheat gluten; (b) glycerol and egg white.
G'
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Fig. 1c shows the evolution of temperature along the mixing process for both gluten and albumen-based bioplastics. A slight increase in temperature, due to mechanical energy dissipation, takes place along the induction stage. After this region, an exponential increase in temperature is observed during gluten processing, which suggests that not only dissipation of mechanical energy is taking place in this material, but also exothermic polymerization reactions of the protein chains. On the contrary, the temperature profile during egg albumen mixing just shows a moderate increase in temperature, although more important than in the previous case, after the induction period and previously to its structural breakdown. Fig. 2 shows the temperature dependence of the linear 0 viscoelasticity functions, storage and loss moduli (G and 00 G ), at a constant frequency of 6.28 rad/s, for different
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these samples dead time operations were carried out, in order not to affect the experimental conditions after the sample extraction. Fig. 1b shows the evolution of torque during the mixing process of egg albumen-based bioplastics. The same three regions in the torque profile previously described may be observed. It is worth pointing out that, in this case, the values of torque reached in the second region are significantly higher than those obtained during processing of glutenbased materials. However, the most important difference is obtained at the third region of the torque profile, where the values of torque suddenly decrease down to zero, due to a dramatic structural breakdown. The blend at this stage becomes granular and heterogeneous. Consequently, only samples obtained at the end of the first region, denoted as G/EW-I, have been processed further.
104 140
T (º C)
Fig. 2. Temperature dependence of the storage ad loss moduli (at 6.28 rad/s) for: (a) wheat gluten-based bioplastics (G/WG-I, after 10 min. processing; G/ WG-II, after 45 min. processing), and (b) egg white-based bioplastics (G/EW-I, after 10 min. processing).
A. Jerez et al. / Journal of Food Engineering 82 (2007) 608–617
0.002 (W/g) Reversing Heat Flow
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samples taken during the mixing process of both glycerol/ gluten (G/WG-I and G/WG-II) and glycerol/egg albumen (G/EW-I) blends. The evolution of the linear viscoelasticity functions with temperature for sample G/WG-I is quite 0 00 complex. Thus, both G and G decrease as temperature increases up to 43 °C. Then, a slight increase in both moduli is noticed as temperature increases up to 60 °C. A further decrease in these linear viscoelasticity functions down to minimum values is observed when temperature 0 is increased up to 100 °C. Between 100 °C and 135 °C, G undergoes a remarkable increase, a fact that may be attributed to protein cross-linking reactions, which occur under severe thermal conditions (Gennadios et al., 1996), 0 although this final increase in G is dampened as temperature increases further. The two first regions described above must be a consequence of the early processing stage at which the sample was taken, because they disappear for sample G/WG-II. Likewise, the initial values of both moduli are much higher for this last sample, as a consequence of its previous thermal and mechanical history (see Fig. 1a and c). Nevertheless, in the high temperature region (around 140 °C), the values of the linear viscoelasticity functions for both samples are quite similar, fact that seems to demonstrate that the linear viscoelastic characteristics of these blends are mainly defined by the protein cross-linking process. Even a more complex behaviour is found for sample G/ EW-I. In a first stage (up to ca. 50 °C), the linear viscoelastic functions, at a constant frequency of 6.28 rad/s, for the glycerol/egg albumen blend undergo a remarkable decrease as temperature increases, reaching similar values of the storage and loss moduli (which indicates an enhancement of the viscous properties of the blend) and a minimum in both linear viscoelasticity functions at ca. 50 °C. Above this temperature, a dramatic increase in both linear viscoelasticity functions takes place up to 65 °C. A further increase in temperature produces a change in the slope of 0 00 00 G and G , yielding a plateau region in G (65–110 °C). 0 On the contrary, a slight increase in G (up to 75 °C) is first noticed. A new increase in temperature (up to 90 °C) yields 0 another dramatic increase in G . In this last region a solidlike behaviour is increasingly strengthened, as can be noticed in Fig. 2b. Furthermore, the final values of both moduli are clearly higher than those for glycerol/gluten blends. The data obtained from oscillatory shear (Fig. 2b) and MDSC measurements (Fig. 3c) suggest that the microstructure of a glycerol/albumen blend results from a superposition of different thermal events. Thus, the reversing heat flow curve shows two glass transition events. The first Tg is found at ca. 54 °C, which is in good agreement with 0 00 the evolution of G and G in this region. Above this temperature, the increase in the linear viscoelastic functions reveals a structural build up due to the beginning of protein denaturation and aggregation, as may be deduced from the non-reversing heat flow curve. This curve shows a broad endothermic event, with a peak located at ca. 86 °C, which
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T (º C) Fig. 3. MDSC thermograms for the different systems studied: (a) G/WG-I (10 min. mixing); (b) G/WG-II (45 min. mixing); (c) G/EW-I (10 min. mixing).
corresponds to the so-called denaturation temperature. Associated with this thermal event a second glass transition is observed in the reversing heat flow curve, located at ca. 88 °C. This gelation process is also demonstrated by a 0 change in the slope in G at ca. 75 °C, and would finish
A. Jerez et al. / Journal of Food Engineering 82 (2007) 608–617
25 °C, during the mixing process (Fig. 1c) and with the increase in the linear viscoelastic functions observed above 40 °C (see Fig. 2a). (Lefebvre, Popineau, & Cornec, 1993) found a similar increase in the linear viscoelastic moduli of gluten/water systems above 55 °C. Some authors have suggested that plasticized-gluten molecular networks involve the dissociation and unfolding of the macromolecules, which allow them to recombine and crosslink through specific linkages (Redl et al., 1999, 2003; Mitchell et al., 1994). The prospective changes in the microstructure of glycerol/egg albumen and glycerol/gluten blends during mixing have been analysed from oscillatory shear data (Fig. 4). From these results, it is apparent that mixing of gluten or egg albumen with glycerol induces significant changes in the microstructure of these blends. Thus, samples G/WGI and G/EW-I display mechanical spectra typical of entanglement networks. However, sample G/EW-I shows a welldefined plateau region and the beginning of the terminal one, whereas the plateau region that appears in the mechanical spectrum of sample G/WG-I, in the experimental frequency window studied, is much closer to the transition region. On the contrary, sample G/WG-II exhibits a mechanical spectrum characteristic of a highly cross-linked network (Gennadios et al., 1996) with much higher values of both moduli. These results may be of a major interest from a technological point of view. Thus, glycerol/egg white blends show a high thermosetting potential at lower temperature (see Fig. 1 and 2) than glycerol/gluten blends, leading to larger values of the linear viscoelastic moduli. In addition, the temperature sweep (Fig. 2 and 3) and oscillatory shear data (Fig. 4) for the glycerol/egg white blend evidence that this blend could be easily handled at temperatures lower than 50 °C, before an eventual thermosetting treatment.
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at about 100 °C, as both rheological and MDSC measurements evidence. In relation to the above-mentioned results, it is worth pointing out that heat-induced gelation of globular proteins has been described as a two-stage process involving first a partial unfolding of protein molecules and the aggregation of unfolded molecules into the gel network during the second stage (Ferry, 1980; Pe´rez & Pilosof, 2004). During this thermal denaturation, the partially unfolded egg white proteins are capable of interacting under favourable conditions to form some complexes. Previous studies on the gelation properties of egg white have demonstrated that, in a mixture of all the egg white proteins, the aggregation of the polypeptides occur in two distinct coagulation temperature ranges (Campbell, Vassilios, & Stephen, 2003). In the first coagulation temperature range, conalbumin and possibly some other proteins were denatured and partially aggregated, whilst coagulation was completed in the second temperature range (Ferry, 1980). The MDSC curves obtained for wheat gluten based samples with different thermomechanical history are shown in Fig. 3a and b. As can be observed, the denaturation temperatures for both glycerol/wheat gluten samples I and II, located at ca. 150 and 160 °C, respectively, are much higher than that found with the glycerol/egg albumen sample previously discussed. A broad glass transition event in the reversing heat flow curve is noticed between 60 and 120 °C for both samples (see Fig. 3a and b), which is consistent with the rheological data obtained (see Fig. 2a). On the other hand, an exothermic event in the non-reversing heat flow curve, located between 40 and 60 °C, is only found in the sample G/WG-I, when the mixing process is at the first stage. The exotherm would result from a new rearrangement of protein molecules and is in good agreement with the increase in temperature observed, above
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Fig. 4. Mechanical spectra for the different systems studied. (a) Glycerol/wheat gluten blends, G/WG-I (10 min. mixing) and G/WG-II (45 min. mixing). (b) Glycerol/egg white blend, G/EW-I (10 min. mixing).
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3.2. Bioplastics by compression moulding
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The results shown above demonstrate that glycerol/protein blends hold a rheological behaviour and a thermosetting potential that would make them suitable for its further thermomechanical processing (extrusion, compression moulding and thermosetting). In this way, egg whitebased bioplastics may be easily manufactured after processing of the blend (extrusion and/or moulding) at a temperature lower than 50 °C, and, afterwards, thermoset in order to obtain a material with the desired mechanical properties. On the contrary, as may be observed in Fig. 2, glycerol/gluten blends require a processing temperature higher than 100 °C to improve their mechanical properties. Fig. 5 shows the DMTA temperature sweep results, at constant frequency, obtained for the different bioplastics studied, after compression moulding and thermosetting of the dough-like materials obtained in the thermomechanical process. The values of the elastic modulus from DMTA temperature sweep tests for gluten-based bioplastics, obtained by compression moulding at a constant pressure of 880 bars and thermosetting temperatures of 90, 120, and 140 °C, are shown in Fig. 5 A and B. An increase in bioplastic thermosetting temperature generally leads both to an increase in the elastic modulus of the bioplastic, although the differences are dampened as temperature increases, and a shift to lower temperatures in the maximum of the loss tangent. However, some differences between the values of the elastic modulus for G/WG-I and G/WG-II samples are 0 noticed in Fig. 5. Initially, the storage modulus (E ) decreases down to minimum values, showing a rubbery-like plateau region at ca. 100 °C. However, a further increase is observed above this temperature, which extent depends on sample characteristics and thermosetting temperature. This last region coincides with the endothermic events described before (see Fig. 3) and would suggest that an apparent thermosetting potential remains after compression and thermal treatment of the sample. In this sense, G/WG-I exhibits a larger thermosetting potential, after heating at 120 and 140 °C, than sample G/WG-II. However, bioplastics with suitable mechanical characteristics cannot be obtained from G/WG-I dough-like samples processed at a thermosetting temperature below 120 °C. Higher thermosetting temperatures yield bioplastics (from G/WG-I samples) 0 with quite similar E values. In order to compare the mechanical properties of these bioplastics to those of a widely used synthetic polymer, the storage modulus values from DMTA temperature sweep tests for a commercial low density polyethylene (LDPE) are also shown in Fig. 5. Gluten-based bioplastics, processed at a thermosetting temperature between 90 and 140 °C, always show lower values of the storage modulus in the whole temperature range studied (Fig. 5a and b). Results from DMTA temperature sweep tests carried out on egg albumen-based bioplastics after compression
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T (ºC) Fig. 5. Dynamic mechanical thermal analysis results for bioplastics, after processing the respective blends at a constant gauge pressure and different temperatures. (a) G/WG-I (10 min. mixing); (b) G/WG-II (45 min. mixing); (c) G/EW-I (10 min. mixing).
moulding, at a constant pressure of 880 bars, and thermosetting at different temperatures (60, 90 and 120 °C) are shown in Fig. 5c. An increase in the values of E’ with thermosetting temperature is also observed. The evolution of elastic modulus during the temperature sweep tests shows a decrease in the range 25–110 °C, whilst a well-defined plateau region is observed above this temperature range. The material thermoset at 60 °C shows maximum and minimum values of the loss tangent and storage modulus,
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respectively, at 86 °C, due to the fact that a certain thermosetting potential remains in the material after processing at the above-mentioned temperature. This further thermosetting process seems to take place during the DMTA temperature sweep test, leading to a rubbery-like plateau region with values quite similar to those shown by bioplastic samples thermoset at higher temperatures (90 and 120 °C). Moreover, the storage modulus values for egg albumenbased bioplastics, thermoset at 90 and 120 °C, are rather similar to those obtained with a commercial LDPE, up to its melting point temperature (ca. 120 °C). Fig. 6 shows DMTA results for sample G/EW-I, after its thermosetting at a constant temperature of 120 °C and different gauge pressures (0, 440 and 880 bars). As can be observed, an initial increase in compression pressure (0– 440 bars) leads to larger storage modulus values. Nevertheless, no significantly differences are noticed after a further increase in the pressure (440–880 bars). In addition, these bioplastics always show qualitatively similar DMTA curves. Thus, a rubbery-like region is clearly observed above 80 °C, which confirms that no additional thermosetting potential remains in the material at processing temperatures above 90 °C. On the other hand, a compression pressure around 440 bars is enough to obtain higher linear viscoelasticity moduli values than those corresponding to LDPE. Moreover, a remarkable change in microstructure is observed at compression pressures around 440 bars, pressure at which the bioplastic becomes transparent, as a result of the combined mechanical and thermal treatments.
3.3. Microstructure As mentioned above, an interesting result obtained after compression moulding of glycerol/albumen blends was the dependency of the visual appearance of the bioplastics on processing conditions. Both temperature and pressure of processing have critical values below which the bioplastics
E' (Pa)
10 8
0.2 10 7
10 6
tanδ
Bioplastic G / EW-I = 0.5; 10' Mixing E' tanδ , 120º C, 0 bar 0.3 , 120º C, 440 bar , 120º C, 880 bar LDPE
10 9
0.1
20
40
60
80
100
120
140
160
180
T (ºC) Fig. 6. Dynamic mechanical thermal analysis results for a bioplastic (from G/EW-I blend) processed at a constant temperature and different gauge pressures.
615
obtained are opaque. On the other hand, transparent glycerol/albumen bioplastics have been obtained at high processing temperature and pressure (T P 60 °C and P P 440 bars). It is well-known that the visual appearance of egg white/ water heat-induced gels depend on the electrostatic repulsions between protein chains, which in turn depend on pH and ionic strength (Oakenfull, Pearce, & Burley, 1997). According to a model for the formation of ovalbumin gel networks reported by Hegg (1982) transparent gels may be obtained when linear aggregates are formed (i.e. at pH values far from the isoelectric point). Conversely, heterogeneous particulate networks formed by random aggregates lead to turbid gels. However, glycerol/egg albumen bioplastics obtained by compression moulding do not seem to follow this pattern and the reasons why egg albumen bioplastics become transparent by increasing pressure are far from being clear. Fig. 7 shows side-by side, and in real time, both tapping mode topography (Fig. 7a and c) and phase images (Fig. 7b and d) for glycerol/albumen samples processed by compression moulding at different pressures. The surface topographies show a rough texture with a preferential alignment direction. However, no differences are noted between samples processed at different pressures. The accompanying cantilever phase angle images show, in both cases (Fig 7b and d), a surface with two different intensity levels (light and dark) irrespective of the changes in local topography, also showing the same direction of alignment. This may be related to the presence of two main components (protein and glycerol) showing different surface mechanical properties, which influence the behaviour of the oscillating cantilever as it taps the surface. Therefore, the areas showing larger phase lag, where protein is probably much more concentrated, should correspond to a higher elasticity level. Thus, the surface area covered by the dark region is in much lower proportion than the light region, which is in good agreement with the composition of the system (Fig. 7b and d). As may be deduced from these images, the light regions result from the contribution of globular-shape units. Some of these units have been arrowed in Fig. 7c and d, which show that these globular particles are not independent but, on the contrary, incorporated into larger aggregates. The size of the arrowed units is of around 80–100 nm. De Groot and De Jongh (2003), using dynamic light scattering analysis, measured ovalbumin aggregates of 85 nm obtained after heat treatment. These authors, assuming a common globular packing of the protein in the aggregate, estimated that the mass of these aggregates would be of the order of 500– 600 kDa, which would correspond to 11–14 molecules of ovalbumin. Therefore, as ovalbumin is in fact the major egg white protein (60–65%), we could speculate with the possibility that the arrowed globules are protein aggregates which associate into larger aggregates. As shown in Fig. 7b, smaller aggregates are detected when the glycerol/albumen sample has been processed at
616
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Fig. 7. AFM micrographs (tapping mode images) for G/EW bioplastics processed at a constant temperature (120 °C) and different gauge pressures. (a) 0 bar gauge pressure topography image; (b) 0 bar gauge pressure phase image; (c) 880 bar gauge pressure topography image; (d) 880 bar gauge pressure phase image.
the minimum pressure. Therefore, an increase in pressure produces larger aggregates, which seem to grow in a preferential direction. This could provide evidence for the formation of higher molecular weight polymers through intermolecular cross-linking. Taking into consideration the thermosetting potentials of either of the two proteins studied, it is possible to design bioplastics with a wide range of mechanical properties by controlling processing conditions and plasticizer content. In addition, these results could be presumably extrapolated to select optimal conditions for other thermomechanical processes such as extrusion and postextrusion operations. 4. Conclusions The mixing process of protein and plasticizer is rather different regarding torque and temperature evolution, and yields bioplastic materials with varied characteristics
depending on the selected protein. Thus, mixing of both glycerol and wheat gluten occurs with an important increase in temperature and suitable viscoelastic dough is obtained only after sufficiently long processing time, whilst egg white protein-based blends should be mixed for shorter times in order to avoid brittleness and obtain viscoelastic dough suitable for further processing. Both, glycerol/gluten and glycerol/albumen blends exhibit rheological properties and thermosetting potentials that make them suitable for further thermomechanical processing (compression moulding, extrusion and thermosetting). However, egg albumen-based blends show higher thermosetting potentials than gluten-based blends. Albumen-based blends can be easily processed (by extrusion and/or moulding) at low temperature (lower than 60 °C), whilst further thermosetting can be performed on the blend in order to obtain materials with the desired mechanical properties.
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An increase in temperature during the compression moulding process yields improved viscoelastic properties for both protein-based bioplastics. In addition, an increase in pressure during albumenbased bioplastic processing favours an increase in aggregate size, which show a clear tendency to form linear aggregates, as has been observed in the AFM images. As a consequence, nearly transparent bioplastics with enhanced mechanical properties are obtained. Acknowledgements This work is part of a research Project sponsored by the MCYT-FEDER Programme (AGL2002-01106) and Junta de Andalucia programme (P06-TEP-02126). The authors gratefully acknowledge its financial support. References ASTM (2001). Standard test method for plastics: dynamic mechanical properties: In flexure (three-point bending). Designation D5023-01. In Annual book of ASTM standards Philadelphia. PA: American Society for Testing and Materials. Boussu, K., Van der Bruggen, B., Volodin, A., Snauwaert, J., Van Haesendonck, C., & Vandecasteele, C. (2005). Roughness and hydrophobicity studies of nanofiltration membranes using different modes of AFM. Journal of Colloid and Interface Science, 286, 632–638. Campbell, L., Vassilios, R., & Stephen, R. E. (2003). Modification of functional properties of egg white proteins. Nahrung Food, 47(6), 369–376. Cherian, G., Gennadios, A., Weller, C., & Chinchoti, P. (1995). Thermomechanical behaviour of wheat gluten films: Effects of sucrose, glycerin, and sorbitol. Cereal Chemistry, 72, 1–6. Cuq, B., Gontard, N., & Guilbert, S. (1999). Effects of thermomoulding process conditions on the properties of agro-materials based on fish myofibrillar proteins. Lebensmittel-Wissenschaft and Technologie, 32, pp. 107, 113, 119. De Graaf, L. A. (2000). Denaturation of proteins from a non-food perspective. Journal of Biotechnology, 79, 299–306. De Groot, J., & De Jongh, H. H. J. (2003). The presence of heat-stable conformers of ovalbumin affects properties of thermally formed aggregates. Protein Engineering, 16, 1035–1040. Dealy, J. M. (1982). Rheometers for molten plastics. New York: Van Nostrand, pp. 255. Domenek, S., Feuilloley, P., Gratraud, J., Morel, M-H., & Guilbert, S. (2004). Biodegradability of wheat gluten based bioplastics. Chemosphere, 54, 551–559. Engel, A., Lyubchenko, Y., & Mu¨ller, D. (1999). Atomic force microscopy: A powerful tool to observe biomolecules at Cork. Trends in Cell Biology, 9, 77–80. Ferry, F. D. (1980). Viscoelastic properties of polymers. New York: Wiley. Gennadios, A. (2002). Protein based films and coatings. New York: CRC press, pp. 66–115. Gennadios, A., Weller, C. L., Hanna, M. A., & Froning, G. W. (1996). Mechanical and barrier properties of egg albumen films. Journal of Food Science, 61, 585–589. Hansma, H. G., Kim, K. J., Laney, D. E., Garcia, R. A., Argaman, M., Allen, M. J., et al. (1997). Properties of biomolecules measured from atomic force microscope images: A review. Journal of Structural Biology, 119, 99–108.
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Hegg, P. (1982). Conditions for the formation of heat-induced gels of some globular food proteins. Journal of Food Science, 47, 1241–1244. Herald, T. J., Gnanasambandam, R., McGuire, B. H., & Hachmeister, K. A. (1995). Degradable wheat gluten films: Preparation, properties and applications. Journal of Food Science, 60, 1147–1156. Irissin-Mangata, J., Ge´rard, B., Bernard, B., & Gontard, N. (2001). New plasticizers for wheat gluten films. European Polymer Journal, 37, 1533–1541. Jerez, A., Partal, P., Martı´nez, I., Gallegos, C., & Guerrero, A. (2005). Rheology and processing of gluten based bioplastics. Biochemical Engineering Journal, 26, 131–138. Lefebvre, J., Popineau, Y., & Cornec, M. (1993). Viscoelastic properties of wheat gluten proteins: Influence of prolamin composition and of temperature. In Seibel, W., Bushuk, W. (Eds.), Gluten proteins. Association of Cereal Research (pp. 180–190). Magonov, S. N., Elings, V., & Whangbo, M. H. (1997). Phase imaging and stiffness in tapping-mode atomic force microscopy. Surface Science, 375, 385–391. Matveev, Y. I., Grinberg, V. Y., & Tolstoguzov, V. B. (2000). The plasticizing effect of water on proteins, polysaccharides and their mixtures. Glassy state of biopolymers, food and seeds. Food Hydrocolloids, 14, 425–437. Mine, Y. (1995). Recent advances in understanding egg white protein functionality. Trends in Food Science Technology, 6, 225–238. Mitchell, J. R., Areas, J. A., Rasul, S., Coloma, P., & Della Valle, G. (1994). Modfications chimiques et texturation des proteines a` faibles teneurs en eau. In P. Coloma & G. Della Valle (Eds.), La caisson extrusion. Technique et documentation (pp. 85–108). Paris: Lavoiser. Oakenfull, D., Pearce, J., & Burley, R. W. (1997). Food proteins and their applications. In S. Damodaran & A. Paraf (Eds.) (pp. 111–142). New York: Marcel Dekker. Pe´rez, O. E., & Pilosof, M. R. A. (2004). Pulsed electric fields effects on the molecular structure and gelation of b-lactoglobulin concentrate and egg white. Food Research International, 23, 102–110. Pouplin, M., Redl, A., & Gontard, N. (1999). Glass transition of wheat gluten plasticized with water, glycerol and sorbitol. Journal of Agricultural and Food Chemistry, 47, 538–543. Redl, A., Morel, M. H., Bonicel, J., Vergnes, B., & Guilbert, S. (1999). Rheological properties of gluten plasticized with glycerol: Dependence on temperatura, glycerol content and mixing conditions. Rheologica Acta, 38, 311–320. Redl, A., Guilbert, S., & Morel, M. H. (2003). Heat and shear mediated polymerisation of plasticized wheat gluten protein upon mixing.. Journal of Cereal Science, 38, 105–114. Rosa-Zeiser, A., Weilandt, E., Hild, S., & Marti, O. (1997). The simultaneous measurement of elastic, electrostatic and adhesive properties by scanning force microscopy: Pulsed-force mode operation. Measurement Science and Technology, 8, 1333–1338. Sheiko, S. S., & Mo¨ller, M. (2001). Visualization of macromolecules. A first step to manipulation and controlled response. Chemistry Review, 101, 4099–4123. Silva, L. P. (2002). Atomic force microscopy and proteins. Protein Peptide Letters, 9, 117–125. Van der Placken, I., Van Loey, A., & Hendrickx, M. E. (2005). Combined effect of high pressure and temperature on selected properties of egg white proteins. Innovative Food Science and Emerging Technologies, 6(1), 11–20. Wahl, K. J., Stepnowski, S. V., & Unertl, W. N. (1998). Viscoelastic effects in nanometer-scale contacts under shear. Tribology Letters, 5, 103–107. Welti-Chanes, J., Barbosa-Ca´novas, G., & Aguilera, J. M. (2002). Engineering and Food for the 21st Century. New York: CRC Press. Yamauchi, A., & Yamauchi, K. (1997). Novel proteinous microcapsules from wool keratins. Colloids and Surfaces B: Biointerfaces, 9, 117–119.
Egg white-based bioplastics developed by thermomechanical processing A. Jerez a, P. Partal a,*, I. Martı´nez a, C. Gallegos a, A. Guerrero b a
Departamento de Ingenierı´a Quı´mica, Facultad de Ciencias Experimentales, Campus del Carmen, Universidad de Huelva, 21071 Huelva, Spain b Departamento de Ingenierı´a Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, 41012 Sevilla, Spain Received 14 September 2006; received in revised form 18 January 2007; accepted 11 March 2007 Available online 20 March 2007
Abstract The development of new protein-based biomaterials has been receiving increasing interest in the last few years. This paper is focused on the development of new bioplastic materials, based on wheat gluten and egg white proteins, manufactured by direct mixing of proteins with a plasticizer, glycerol, and, eventually, a thermal and moulding process which shapes the material and gives them suitable mechanical properties to be used as substitutive materials of synthetic polymers for definite applications. These rheological properties have been evaluated by shear rheology and dynamic mechanical thermal analysis on both intermediate process materials and final bioplastics. Moreover, the microstructure of the samples has been characterized by atomic force microscopy and modulated differential scanning calorimetry. Glycerol–protein mixtures have been proved to hold suitable rhelogical behaviour and thermosetting potential for further processing, by means of a compression-moulding process, to obtain bioplastics. Glycerol/egg white blends (0.5 ratio) can be more easily processed, because of their rheological behaviour at ca. 50 °C. On the contrary, glycerol/wheat gluten blends (0.5 ratio) need longer mixing times and more severe thermosetting conditions, although lower values of the bioplastic storage modulus are obtained in the whole range of temperature studied. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Egg white; Wheat gluten; Bioplastics; Rheology; Viscoelasticity; Processing
1. Introduction Biopolymers from agricultural sources are becoming a suitable alternative not only as biodegradable films, suitable for food packaging, but also as plastic stuffs which require improved mechanical properties. Proteins, lipids and polysaccharides have been used as biopolymer sources for many years. It is well-known that a wide variety of proteins are produced at huge scale for instance, wheat gluten, soy and pea proteins from vegetable resources; egg proteins, fish myofibrillars and wool keratin proteins from animal resources (Cuq, Gontard, & Guilbert, 1999; De Graaf, 2000; Domenek, Feuilloley, Gratraud, Morel, & Guilbert, 2004; Irissin-Mangata, Ge´rard, Bernard, & Gontard, 2001; Mine, 1995; Yamauchi & Yamauchi, 1997). In addi*
Corresponding author. E-mail address: [email protected] (P. Partal).
0260-8774/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2007.03.020
tion, biopolymers have the advantage of biodegradability, which adds an extra value to materials developed from them. Thus, protein-based biomaterials degradation rates have been proved to be among the rates of fast-degrading polymers, completely degrading in 50 days when buried in farmland soils (Domenek et al., 2004). Proteins used for food applications should be in their native form (non-denaturated state) in order to keep their functional properties. However, for non-edible applications, protein denaturation becomes an additional variable for the design of new materials, which properties may change depending on the denaturation degree of the proteins that conform them (De Graaf, 2000). Hen egg white proteins (albumen) have been often used as ingredients in food processing for their unique functional properties, such as gelling, foaming, heat setting and binding adhesion. Up to now, the structural and functional properties of egg white proteins have been studied
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mainly focusing on the contribution of the major egg white protein, the ovalbumin. Ovalbumin constitutes over half of the egg white albumen. It is a monomeric phosphoglycoprotein and the only protein containing free sulfhydril groups, four, which are buried in the protein core. Heatinduced denaturation of ovalbumin results in the external exposure of these sulfhydril groups, accompanied by a decrease in the total sulfhydril content, due to the oxidation of SH groups to disulfide bonds (Van der Placken, Van Loey, & Hendrickx, 2005). A plasticizer is a film component, required to overcome film brittleness and helping to avoid chipping and cracking of films during subsequent handling and storage. Plasticizers are molecules with low molecular weight and low volatility, which modify the three-dimensional structure of proteins (Matveev, Grinberg, & Tolstoguzov, 2000). These plasticizers have a general behaviour that follows the Couchmann–Karasz relation using a heat capacity (DCp) value of 0.4 J/g K for wheat gluten (Pouplin, Redl, & Gontard, 1999). Comparing the plasticizer efficiency on a weight basis, a similar plastification has been obtained with plasticizers of different molecular structure (hydroxyl or amino groups). The plasticizing efficiency has been reported to be generally proportional to the molecular weight and inversely proportional to the percent of hydrophilic groups of the plasticizer (Welti-Chanes, BarbosaCa´novas, & Aguilera, 2002). Processing of films, coatings or other materials based on polymers derived from proteins requires three main steps: breaking of intermolecular bonds (non-covalent and covalent, if necessary) that stabilize the polymers in their native forms, by using chemical or physical rupturing agents; arranging and orienting mobile polymer chains into the desired shape; and, finally, allowing the formation of new intermolecular bonds and interactions to stabilize the three-dimensional network. The shape obtained in the second step is mainly maintained by eliminating the agents used to break the intermolecular bonds in the first step. Active sites for bond formation become free and close enough to each other to create new interactions, hydrogen bonds, hydrophobic interactions and disulfide bonds, forming a new three-dimensional network (Gennadios, 2002). The casting method, or physico-chemical method, for film processing is based on the above mentioned three steps, using a chemical reactant to disrupt disulfide bonds, dispersing, solubilizing proteins and finally drying it. Other authors, who have studied film formation by the casting method, have processed 50 lm thick samples, obtaining good quality films which could be used for food packaging in view of the properties they possess such as opacity, water vapour, oxygen and carbon dioxide permeability, and mechanical properties. However, the use of these films for other applications where they could substitute synthetic polymers is quite limited (Cherian, Gennadios, Weller, & Chinchoti, 1995; Herald, Gnanasambandam, McGuire, & Hachmeister, 1995).
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Another way of processing biomaterials is the mechanical method, or thermoplastic processing, which consists of mixing proteins and plasticizer to obtain a dough-like material. Therefore, the extrusion of proteins is, in general, only possible in a limited window of operating conditions and the material properties of the extrudates depend on the processing conditions in a complex way (Mitchell, Areas, Rasul, Coloma, & Della Valle, 1994; Redl, Morel, Bonicel, Vergnes, & Guilbert, 1999; Redl, Guilbert, & Morel, 2003). At this moment, most of the procedures for manufacturing egg white proteins films are based on the casting method (Gennadios, Weller, Hanna, & Froning, 1996). On the contrary, the use of a thermo-mechanical method to obtain an egg white-based bioplastic has not been previously investigated. This new method makes the bioplastic processing easier and may have an interesting industrial potential. As a result, the overall objective of this work was to obtain bioplastics from egg white proteins by means of a combined compression moulding/thermosetting procedure. This paper focuses on the effects that processing and a further thermal treatment exert on the thermo-mechanical properties and microstructure of the biomaterials obtained, and how they can be modified if the denaturation degree of the protein changes. The results obtained have been compared with those obtained from wheat gluten based bioplastics. 2. Materials and methods Wheat gluten was provided by RIBA S.A. Spray-dried egg white albumen was provided by OVOSEC S.A. Some compositional characteristics of these protein concentrates used as raw materials in this work are shown in Table 1. Glycerol, from Panreac Quı´mica, S.A. (Spain), was used as protein plasticizer. The thermo-mechanical processing was carried out in a torque-rheometer (Polylab, Haake, Germany) for the simulation of industrial processes in the lab or pilot plants, by means of the use of different modular tools. The mixer tool (Rheomix 3000p) enables the batch testing of many highly viscous substances and consists of a batch mixer fitted with two counter-rotating rollers, turning with different angular velocities (ratio 3:2). A detailed description of a common torque-rheometer can be found elsewhere (Dealy, 1982). Protein and glycerol were directly added in the mixing chamber before starting the mixing tests. Both torque Table 1 Some compositional characteristics of the protein concentrates used as raw materials in this work
Protein content (%) Lipids (%) Ashes (%) Moisture (%) Reducing sugars (%) pH (10% solution)
Wheat gluten (WG)
Egg white (EW)
83 1.5–2 0.7–0.8 8 – 6.88
73 – 6 8 0.1 7.10
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and temperature have been recorded during the mixing process. The mixing chamber can be considered adiabatic, as it was not cooled. The volume of the chamber (310 cm3) was filled with approximately 245 g of sample, corresponding to 80% filling ratio. The plasticizer concentration was 0.5 g glycerol/g protein for both gluten and albumen. The mixing process was always carried out at 50 rpm. Compression-moulded biomaterials were prepared by compressing the dough-like materials obtained after the mixing process at different gauge pressures, in a 50 10 3 mm mould. The equipment used to carry out this process was developed by our own laboratory facilities, consisting in a two electrical heated plates, with electronic temperature control, and a hydraulic pressure system. The compression-moulding process was carried out at three different temperatures 90, 120, and 140 °C, for wheat gluten bioplastics, and 60, 90 and 120 °C, for egg albumen bioplastics, during 10 min. Theses temperatures were above the maximum temperature reached during the mixing process and below the temperature at which the degradation of the blends takes place. Frequency sweep tests, from 0.01 to 100 rad/s, in oscillatory shear, at a constant stress within the linear viscoelastic region and a temperature of 25 °C, were conducted in a controlled stress rheometer Rheoscope (Haake, Germany), using a serrated parallel plate geometry, 20 mm diameter and 1–1.2 mm gap. Previously, stress sweep tests, at 6.28 rad/s, were carried out in order to identify the linear viscoelastic region of these materials. Temperature sweep tests, from 25 to 170 °C, were conducted in a controlled strain rheometer ARES (Rheometric Scientific, USA), using a serrated parallel plate geometry, 25 mm diameter and 1–1.2 mm gap. Measurements were performed at constant frequency (6.28 rad/s) and strain (1%, which was always within the linear viscoelastic region), selecting a 2 °C/min. temperature ramp. DMTA experiments were done with a Seiko DMS 6100 (Seiko Instruments, Japan), using 50 10 3 mm samples in double cantilever bending mode, according to the ASTM standard method D5023-01 (ASTM, 2001). All the experiments were carried out at constant frequency (1 Hz) and strain (within the linear viscoelastic region). The selected temperature ramp was 2 °C/min. MDSC experiments were performed with a Q100 (TA Instruments, USA), using 10–20 mg samples, in hermetic aluminium pans. An oscillation period of 60 s, with an amplitude of ±0.5 °C, and a heating rate of 5 °C/min, were selected. The sample was purged with a nitrogen flow of 50 ml/min. All the rheological and calorimetrical measurements were replicated at least three times. The microstructural characterization of the samples was carried out by means of atomic force microscopy (AFM) with a multimode AFM (Digital Instruments, Veeco Metrology Group; Inc., Santa Barbara, CA) connected to a Nanoscope_IV scanning probe microscope controller (Digital Instruments, Veeco Metrology Group Inc., Santa
Barbara, CA). All images were acquired in tapping mode using Veeco NanoprobeTM tips (Veeco Metrology Group Inc., Santa Barbara, CA). Atomic force microscopy (AFM) has arisen as one of the most powerful tools for determining the surface topography of biomacromolecules at nanometer or even subnanometer resolution (Engel, Lyubchenko, & Mu¨ller, 1999; Hansma et al., 1997; Silva, 2002). Furthermore, AFM images of biomaterials can give not only structural information but also knowledge on some materials properties of the sample. In some cases, new imaging modes generate AFM images that reflect material properties of the sample surface. An excellent example is the phase mode imaging, being a powerful extension of tapping mode AFM, which provides nanometer-scale information about surface structure and properties often not revealed by other scanning probe microscopy techniques. Phase imaging, which is simultaneously monitored with topography data, measures the phase shift between the oscillation driving the tip and the oscillation produced by the tip as it interacts with features on the sample surface. The phase lag is very sensitive to many material properties, such as variations in composition, adhesion, friction and viscoelasticity (Rosa-Zeiser, Weilandt, Hild, & Marti, 1997; Sheiko & Mo¨ller, 2001; Wahl, Stepnowski, & Unertl, 1998), and may show patterns of stiffness and/or hydrophobicity on the sample surface (Boussu et al., 2005; Magonov, Elings, & Whangbo, 1997). However, the interpretation of phase images is still at an early stage, so that there is currently no simple correlation between phase contrast and a single material property. 3. Results and discussion 3.1. Mechanical processing Processing of bioplastics implies the intimate mixing of plasticizers and proteins. Fig. 1 shows the evolution of both torque and temperature during this mixing process for glycerol/gluten (Fig. 1a) and glycerol/egg albumen (Fig. 1b) blends (plasticizer/protein ratio: 0.5). Three different regions may be observed (Fig. 1a). The first region, where no significant increase in torque is observed, corresponds to an induction period that takes place at low mixing times. In this first stage of processing, a dough-like material is obtained, which may be suitable for further thermo-mechanical treatments. In the second region, torque undergoes an exponential increase with mixing time up to a maximum value. Finally, an apparent torque decay is observed in the last region. Previous results obtained by the authors showed an important dependence of this torque profile on mixing conditions (Jerez, Partal, Martı´nez, Gallegos, & Guerrero, 2005). Two samples at different stages of the mixing process were selected in order to investigate the effect of the thermo-mechanical processing. The first one, denoted as sample G/WG-I, was taken out at the end of the first stage (after 10 min. mixing), whilst sample G/WG-II, which corresponds to the torque decay stage, was taken after 45 min. of processing. For each one of
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a
611
b
200
80
200
C
70
0.5 G/WG 0.5 G/EW
60
T (º C)
160
160
50 40
20
0
10
20
30
40
120
50
t (min)
80
80
II
0.5 G/WG 0.5 G/EW
40
40
I 0
M (N·m)
M (N·m)
30
120
0
I
10
20
30
40
5
10
t (min)
0 20
15
t (min)
Fig. 1. Evolution of torque (a and b) and temperature (c) during the mixing process of proteins and plasticizers (plasticizer/protein ratio: 0.5). (a) glycerol and wheat gluten; (b) glycerol and egg white.
G'
G', G'' (Pa)
a
G'' , , ,
106
Fig. 1c shows the evolution of temperature along the mixing process for both gluten and albumen-based bioplastics. A slight increase in temperature, due to mechanical energy dissipation, takes place along the induction stage. After this region, an exponential increase in temperature is observed during gluten processing, which suggests that not only dissipation of mechanical energy is taking place in this material, but also exothermic polymerization reactions of the protein chains. On the contrary, the temperature profile during egg albumen mixing just shows a moderate increase in temperature, although more important than in the previous case, after the induction period and previously to its structural breakdown. Fig. 2 shows the temperature dependence of the linear 0 viscoelasticity functions, storage and loss moduli (G and 00 G ), at a constant frequency of 6.28 rad/s, for different
b
0.5 G/WG-I, 10' mixing 0.5 G/WG-II, 45' mixing 0.5 G/EW-I, 10' mixing
106
105
105
104 20
40
60
80 100 120 140 160
T (º C)
20
40
60
80
100
120
G', G'' (Pa)
these samples dead time operations were carried out, in order not to affect the experimental conditions after the sample extraction. Fig. 1b shows the evolution of torque during the mixing process of egg albumen-based bioplastics. The same three regions in the torque profile previously described may be observed. It is worth pointing out that, in this case, the values of torque reached in the second region are significantly higher than those obtained during processing of glutenbased materials. However, the most important difference is obtained at the third region of the torque profile, where the values of torque suddenly decrease down to zero, due to a dramatic structural breakdown. The blend at this stage becomes granular and heterogeneous. Consequently, only samples obtained at the end of the first region, denoted as G/EW-I, have been processed further.
104 140
T (º C)
Fig. 2. Temperature dependence of the storage ad loss moduli (at 6.28 rad/s) for: (a) wheat gluten-based bioplastics (G/WG-I, after 10 min. processing; G/ WG-II, after 45 min. processing), and (b) egg white-based bioplastics (G/EW-I, after 10 min. processing).
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0.002 (W/g) Reversing Heat Flow
Total Heat Flow, Nonreversing Heat Flow 0.02 (W/g)
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samples taken during the mixing process of both glycerol/ gluten (G/WG-I and G/WG-II) and glycerol/egg albumen (G/EW-I) blends. The evolution of the linear viscoelasticity functions with temperature for sample G/WG-I is quite 0 00 complex. Thus, both G and G decrease as temperature increases up to 43 °C. Then, a slight increase in both moduli is noticed as temperature increases up to 60 °C. A further decrease in these linear viscoelasticity functions down to minimum values is observed when temperature 0 is increased up to 100 °C. Between 100 °C and 135 °C, G undergoes a remarkable increase, a fact that may be attributed to protein cross-linking reactions, which occur under severe thermal conditions (Gennadios et al., 1996), 0 although this final increase in G is dampened as temperature increases further. The two first regions described above must be a consequence of the early processing stage at which the sample was taken, because they disappear for sample G/WG-II. Likewise, the initial values of both moduli are much higher for this last sample, as a consequence of its previous thermal and mechanical history (see Fig. 1a and c). Nevertheless, in the high temperature region (around 140 °C), the values of the linear viscoelasticity functions for both samples are quite similar, fact that seems to demonstrate that the linear viscoelastic characteristics of these blends are mainly defined by the protein cross-linking process. Even a more complex behaviour is found for sample G/ EW-I. In a first stage (up to ca. 50 °C), the linear viscoelastic functions, at a constant frequency of 6.28 rad/s, for the glycerol/egg albumen blend undergo a remarkable decrease as temperature increases, reaching similar values of the storage and loss moduli (which indicates an enhancement of the viscous properties of the blend) and a minimum in both linear viscoelasticity functions at ca. 50 °C. Above this temperature, a dramatic increase in both linear viscoelasticity functions takes place up to 65 °C. A further increase in temperature produces a change in the slope of 0 00 00 G and G , yielding a plateau region in G (65–110 °C). 0 On the contrary, a slight increase in G (up to 75 °C) is first noticed. A new increase in temperature (up to 90 °C) yields 0 another dramatic increase in G . In this last region a solidlike behaviour is increasingly strengthened, as can be noticed in Fig. 2b. Furthermore, the final values of both moduli are clearly higher than those for glycerol/gluten blends. The data obtained from oscillatory shear (Fig. 2b) and MDSC measurements (Fig. 3c) suggest that the microstructure of a glycerol/albumen blend results from a superposition of different thermal events. Thus, the reversing heat flow curve shows two glass transition events. The first Tg is found at ca. 54 °C, which is in good agreement with 0 00 the evolution of G and G in this region. Above this temperature, the increase in the linear viscoelastic functions reveals a structural build up due to the beginning of protein denaturation and aggregation, as may be deduced from the non-reversing heat flow curve. This curve shows a broad endothermic event, with a peak located at ca. 86 °C, which
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T (º C) Fig. 3. MDSC thermograms for the different systems studied: (a) G/WG-I (10 min. mixing); (b) G/WG-II (45 min. mixing); (c) G/EW-I (10 min. mixing).
corresponds to the so-called denaturation temperature. Associated with this thermal event a second glass transition is observed in the reversing heat flow curve, located at ca. 88 °C. This gelation process is also demonstrated by a 0 change in the slope in G at ca. 75 °C, and would finish
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25 °C, during the mixing process (Fig. 1c) and with the increase in the linear viscoelastic functions observed above 40 °C (see Fig. 2a). (Lefebvre, Popineau, & Cornec, 1993) found a similar increase in the linear viscoelastic moduli of gluten/water systems above 55 °C. Some authors have suggested that plasticized-gluten molecular networks involve the dissociation and unfolding of the macromolecules, which allow them to recombine and crosslink through specific linkages (Redl et al., 1999, 2003; Mitchell et al., 1994). The prospective changes in the microstructure of glycerol/egg albumen and glycerol/gluten blends during mixing have been analysed from oscillatory shear data (Fig. 4). From these results, it is apparent that mixing of gluten or egg albumen with glycerol induces significant changes in the microstructure of these blends. Thus, samples G/WGI and G/EW-I display mechanical spectra typical of entanglement networks. However, sample G/EW-I shows a welldefined plateau region and the beginning of the terminal one, whereas the plateau region that appears in the mechanical spectrum of sample G/WG-I, in the experimental frequency window studied, is much closer to the transition region. On the contrary, sample G/WG-II exhibits a mechanical spectrum characteristic of a highly cross-linked network (Gennadios et al., 1996) with much higher values of both moduli. These results may be of a major interest from a technological point of view. Thus, glycerol/egg white blends show a high thermosetting potential at lower temperature (see Fig. 1 and 2) than glycerol/gluten blends, leading to larger values of the linear viscoelastic moduli. In addition, the temperature sweep (Fig. 2 and 3) and oscillatory shear data (Fig. 4) for the glycerol/egg white blend evidence that this blend could be easily handled at temperatures lower than 50 °C, before an eventual thermosetting treatment.
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at about 100 °C, as both rheological and MDSC measurements evidence. In relation to the above-mentioned results, it is worth pointing out that heat-induced gelation of globular proteins has been described as a two-stage process involving first a partial unfolding of protein molecules and the aggregation of unfolded molecules into the gel network during the second stage (Ferry, 1980; Pe´rez & Pilosof, 2004). During this thermal denaturation, the partially unfolded egg white proteins are capable of interacting under favourable conditions to form some complexes. Previous studies on the gelation properties of egg white have demonstrated that, in a mixture of all the egg white proteins, the aggregation of the polypeptides occur in two distinct coagulation temperature ranges (Campbell, Vassilios, & Stephen, 2003). In the first coagulation temperature range, conalbumin and possibly some other proteins were denatured and partially aggregated, whilst coagulation was completed in the second temperature range (Ferry, 1980). The MDSC curves obtained for wheat gluten based samples with different thermomechanical history are shown in Fig. 3a and b. As can be observed, the denaturation temperatures for both glycerol/wheat gluten samples I and II, located at ca. 150 and 160 °C, respectively, are much higher than that found with the glycerol/egg albumen sample previously discussed. A broad glass transition event in the reversing heat flow curve is noticed between 60 and 120 °C for both samples (see Fig. 3a and b), which is consistent with the rheological data obtained (see Fig. 2a). On the other hand, an exothermic event in the non-reversing heat flow curve, located between 40 and 60 °C, is only found in the sample G/WG-I, when the mixing process is at the first stage. The exotherm would result from a new rearrangement of protein molecules and is in good agreement with the increase in temperature observed, above
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Fig. 4. Mechanical spectra for the different systems studied. (a) Glycerol/wheat gluten blends, G/WG-I (10 min. mixing) and G/WG-II (45 min. mixing). (b) Glycerol/egg white blend, G/EW-I (10 min. mixing).
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3.2. Bioplastics by compression moulding
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The results shown above demonstrate that glycerol/protein blends hold a rheological behaviour and a thermosetting potential that would make them suitable for its further thermomechanical processing (extrusion, compression moulding and thermosetting). In this way, egg whitebased bioplastics may be easily manufactured after processing of the blend (extrusion and/or moulding) at a temperature lower than 50 °C, and, afterwards, thermoset in order to obtain a material with the desired mechanical properties. On the contrary, as may be observed in Fig. 2, glycerol/gluten blends require a processing temperature higher than 100 °C to improve their mechanical properties. Fig. 5 shows the DMTA temperature sweep results, at constant frequency, obtained for the different bioplastics studied, after compression moulding and thermosetting of the dough-like materials obtained in the thermomechanical process. The values of the elastic modulus from DMTA temperature sweep tests for gluten-based bioplastics, obtained by compression moulding at a constant pressure of 880 bars and thermosetting temperatures of 90, 120, and 140 °C, are shown in Fig. 5 A and B. An increase in bioplastic thermosetting temperature generally leads both to an increase in the elastic modulus of the bioplastic, although the differences are dampened as temperature increases, and a shift to lower temperatures in the maximum of the loss tangent. However, some differences between the values of the elastic modulus for G/WG-I and G/WG-II samples are 0 noticed in Fig. 5. Initially, the storage modulus (E ) decreases down to minimum values, showing a rubbery-like plateau region at ca. 100 °C. However, a further increase is observed above this temperature, which extent depends on sample characteristics and thermosetting temperature. This last region coincides with the endothermic events described before (see Fig. 3) and would suggest that an apparent thermosetting potential remains after compression and thermal treatment of the sample. In this sense, G/WG-I exhibits a larger thermosetting potential, after heating at 120 and 140 °C, than sample G/WG-II. However, bioplastics with suitable mechanical characteristics cannot be obtained from G/WG-I dough-like samples processed at a thermosetting temperature below 120 °C. Higher thermosetting temperatures yield bioplastics (from G/WG-I samples) 0 with quite similar E values. In order to compare the mechanical properties of these bioplastics to those of a widely used synthetic polymer, the storage modulus values from DMTA temperature sweep tests for a commercial low density polyethylene (LDPE) are also shown in Fig. 5. Gluten-based bioplastics, processed at a thermosetting temperature between 90 and 140 °C, always show lower values of the storage modulus in the whole temperature range studied (Fig. 5a and b). Results from DMTA temperature sweep tests carried out on egg albumen-based bioplastics after compression
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T (ºC) Fig. 5. Dynamic mechanical thermal analysis results for bioplastics, after processing the respective blends at a constant gauge pressure and different temperatures. (a) G/WG-I (10 min. mixing); (b) G/WG-II (45 min. mixing); (c) G/EW-I (10 min. mixing).
moulding, at a constant pressure of 880 bars, and thermosetting at different temperatures (60, 90 and 120 °C) are shown in Fig. 5c. An increase in the values of E’ with thermosetting temperature is also observed. The evolution of elastic modulus during the temperature sweep tests shows a decrease in the range 25–110 °C, whilst a well-defined plateau region is observed above this temperature range. The material thermoset at 60 °C shows maximum and minimum values of the loss tangent and storage modulus,
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respectively, at 86 °C, due to the fact that a certain thermosetting potential remains in the material after processing at the above-mentioned temperature. This further thermosetting process seems to take place during the DMTA temperature sweep test, leading to a rubbery-like plateau region with values quite similar to those shown by bioplastic samples thermoset at higher temperatures (90 and 120 °C). Moreover, the storage modulus values for egg albumenbased bioplastics, thermoset at 90 and 120 °C, are rather similar to those obtained with a commercial LDPE, up to its melting point temperature (ca. 120 °C). Fig. 6 shows DMTA results for sample G/EW-I, after its thermosetting at a constant temperature of 120 °C and different gauge pressures (0, 440 and 880 bars). As can be observed, an initial increase in compression pressure (0– 440 bars) leads to larger storage modulus values. Nevertheless, no significantly differences are noticed after a further increase in the pressure (440–880 bars). In addition, these bioplastics always show qualitatively similar DMTA curves. Thus, a rubbery-like region is clearly observed above 80 °C, which confirms that no additional thermosetting potential remains in the material at processing temperatures above 90 °C. On the other hand, a compression pressure around 440 bars is enough to obtain higher linear viscoelasticity moduli values than those corresponding to LDPE. Moreover, a remarkable change in microstructure is observed at compression pressures around 440 bars, pressure at which the bioplastic becomes transparent, as a result of the combined mechanical and thermal treatments.
3.3. Microstructure As mentioned above, an interesting result obtained after compression moulding of glycerol/albumen blends was the dependency of the visual appearance of the bioplastics on processing conditions. Both temperature and pressure of processing have critical values below which the bioplastics
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obtained are opaque. On the other hand, transparent glycerol/albumen bioplastics have been obtained at high processing temperature and pressure (T P 60 °C and P P 440 bars). It is well-known that the visual appearance of egg white/ water heat-induced gels depend on the electrostatic repulsions between protein chains, which in turn depend on pH and ionic strength (Oakenfull, Pearce, & Burley, 1997). According to a model for the formation of ovalbumin gel networks reported by Hegg (1982) transparent gels may be obtained when linear aggregates are formed (i.e. at pH values far from the isoelectric point). Conversely, heterogeneous particulate networks formed by random aggregates lead to turbid gels. However, glycerol/egg albumen bioplastics obtained by compression moulding do not seem to follow this pattern and the reasons why egg albumen bioplastics become transparent by increasing pressure are far from being clear. Fig. 7 shows side-by side, and in real time, both tapping mode topography (Fig. 7a and c) and phase images (Fig. 7b and d) for glycerol/albumen samples processed by compression moulding at different pressures. The surface topographies show a rough texture with a preferential alignment direction. However, no differences are noted between samples processed at different pressures. The accompanying cantilever phase angle images show, in both cases (Fig 7b and d), a surface with two different intensity levels (light and dark) irrespective of the changes in local topography, also showing the same direction of alignment. This may be related to the presence of two main components (protein and glycerol) showing different surface mechanical properties, which influence the behaviour of the oscillating cantilever as it taps the surface. Therefore, the areas showing larger phase lag, where protein is probably much more concentrated, should correspond to a higher elasticity level. Thus, the surface area covered by the dark region is in much lower proportion than the light region, which is in good agreement with the composition of the system (Fig. 7b and d). As may be deduced from these images, the light regions result from the contribution of globular-shape units. Some of these units have been arrowed in Fig. 7c and d, which show that these globular particles are not independent but, on the contrary, incorporated into larger aggregates. The size of the arrowed units is of around 80–100 nm. De Groot and De Jongh (2003), using dynamic light scattering analysis, measured ovalbumin aggregates of 85 nm obtained after heat treatment. These authors, assuming a common globular packing of the protein in the aggregate, estimated that the mass of these aggregates would be of the order of 500– 600 kDa, which would correspond to 11–14 molecules of ovalbumin. Therefore, as ovalbumin is in fact the major egg white protein (60–65%), we could speculate with the possibility that the arrowed globules are protein aggregates which associate into larger aggregates. As shown in Fig. 7b, smaller aggregates are detected when the glycerol/albumen sample has been processed at
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Fig. 7. AFM micrographs (tapping mode images) for G/EW bioplastics processed at a constant temperature (120 °C) and different gauge pressures. (a) 0 bar gauge pressure topography image; (b) 0 bar gauge pressure phase image; (c) 880 bar gauge pressure topography image; (d) 880 bar gauge pressure phase image.
the minimum pressure. Therefore, an increase in pressure produces larger aggregates, which seem to grow in a preferential direction. This could provide evidence for the formation of higher molecular weight polymers through intermolecular cross-linking. Taking into consideration the thermosetting potentials of either of the two proteins studied, it is possible to design bioplastics with a wide range of mechanical properties by controlling processing conditions and plasticizer content. In addition, these results could be presumably extrapolated to select optimal conditions for other thermomechanical processes such as extrusion and postextrusion operations. 4. Conclusions The mixing process of protein and plasticizer is rather different regarding torque and temperature evolution, and yields bioplastic materials with varied characteristics
depending on the selected protein. Thus, mixing of both glycerol and wheat gluten occurs with an important increase in temperature and suitable viscoelastic dough is obtained only after sufficiently long processing time, whilst egg white protein-based blends should be mixed for shorter times in order to avoid brittleness and obtain viscoelastic dough suitable for further processing. Both, glycerol/gluten and glycerol/albumen blends exhibit rheological properties and thermosetting potentials that make them suitable for further thermomechanical processing (compression moulding, extrusion and thermosetting). However, egg albumen-based blends show higher thermosetting potentials than gluten-based blends. Albumen-based blends can be easily processed (by extrusion and/or moulding) at low temperature (lower than 60 °C), whilst further thermosetting can be performed on the blend in order to obtain materials with the desired mechanical properties.
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An increase in temperature during the compression moulding process yields improved viscoelastic properties for both protein-based bioplastics. In addition, an increase in pressure during albumenbased bioplastic processing favours an increase in aggregate size, which show a clear tendency to form linear aggregates, as has been observed in the AFM images. As a consequence, nearly transparent bioplastics with enhanced mechanical properties are obtained. Acknowledgements This work is part of a research Project sponsored by the MCYT-FEDER Programme (AGL2002-01106) and Junta de Andalucia programme (P06-TEP-02126). The authors gratefully acknowledge its financial support. References ASTM (2001). Standard test method for plastics: dynamic mechanical properties: In flexure (three-point bending). Designation D5023-01. In Annual book of ASTM standards Philadelphia. PA: American Society for Testing and Materials. Boussu, K., Van der Bruggen, B., Volodin, A., Snauwaert, J., Van Haesendonck, C., & Vandecasteele, C. (2005). Roughness and hydrophobicity studies of nanofiltration membranes using different modes of AFM. Journal of Colloid and Interface Science, 286, 632–638. Campbell, L., Vassilios, R., & Stephen, R. E. (2003). Modification of functional properties of egg white proteins. Nahrung Food, 47(6), 369–376. Cherian, G., Gennadios, A., Weller, C., & Chinchoti, P. (1995). Thermomechanical behaviour of wheat gluten films: Effects of sucrose, glycerin, and sorbitol. Cereal Chemistry, 72, 1–6. Cuq, B., Gontard, N., & Guilbert, S. (1999). Effects of thermomoulding process conditions on the properties of agro-materials based on fish myofibrillar proteins. Lebensmittel-Wissenschaft and Technologie, 32, pp. 107, 113, 119. De Graaf, L. A. (2000). Denaturation of proteins from a non-food perspective. Journal of Biotechnology, 79, 299–306. De Groot, J., & De Jongh, H. H. J. (2003). The presence of heat-stable conformers of ovalbumin affects properties of thermally formed aggregates. Protein Engineering, 16, 1035–1040. Dealy, J. M. (1982). Rheometers for molten plastics. New York: Van Nostrand, pp. 255. Domenek, S., Feuilloley, P., Gratraud, J., Morel, M-H., & Guilbert, S. (2004). Biodegradability of wheat gluten based bioplastics. Chemosphere, 54, 551–559. Engel, A., Lyubchenko, Y., & Mu¨ller, D. (1999). Atomic force microscopy: A powerful tool to observe biomolecules at Cork. Trends in Cell Biology, 9, 77–80. Ferry, F. D. (1980). Viscoelastic properties of polymers. New York: Wiley. Gennadios, A. (2002). Protein based films and coatings. New York: CRC press, pp. 66–115. Gennadios, A., Weller, C. L., Hanna, M. A., & Froning, G. W. (1996). Mechanical and barrier properties of egg albumen films. Journal of Food Science, 61, 585–589. Hansma, H. G., Kim, K. J., Laney, D. E., Garcia, R. A., Argaman, M., Allen, M. J., et al. (1997). Properties of biomolecules measured from atomic force microscope images: A review. Journal of Structural Biology, 119, 99–108.
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