Development of albumen/soy biobased plastic materials processed by injection molding

1 Development of albumen/soy biobased plastic materials 1 processed by injection molding 2 M. Félix, J.E. Martín-Alfonso, A. Romero*, A. Guerrero 3 Departamento de Ingeniería Química, Universidad de Sevilla, Facultad de Química, 41012 4 Sevilla, Spain 5 Abstract 6 Biobased plastics from renewable polymers constitute a highly interesting field for 7 relevant industrial applications such as packaging, agriculture, etc., in which 8 thermomechanical techniques (i.e. extrusion, compression molding, etc.) are 9 increasingly being used. In spite of the potentials of injection molding in the 10 manufacture of shaped products it is still scarcely used with biopolymers. This study 11 evaluates injection molding as an alternative to produce biobased materials from 12 blends prepared in a mixing rheometer, using different albumen/soy ratios and glycerol 13 as the plasticizer. Viscoelastic measurements and DSC of protein/glycerol blends were 14 used to select suitable processing conditions. Physicochemical properties of injection- 15 molded probes were characterized through dynamic mechanical thermal analysis, 16 tensile strength, water uptake and transmittance tests. Occurrence of shear-induced 17 effects over mixing was confirmed by extractability analysis of protein concentrates and 18 blends, particularly for soy-based systems. Both proteins and their mixtures yield 19 injection-molded bioplastics, although showing lower mechanical properties than LDPE 20 standards. 21 Keywords: Albumen; Bioplastic; Dynamic Mechanical Thermal Analysis; Soy Protein; 22 Tensile strength test; Transmittance. 23 _______________________ 24 *A. ROMERO 25 Departamento de Ingeniería Química, 26 2 Universidad de Sevilla, Facultad de Química, 27 41012 Sevilla (Spain) 28 E-mail: [email protected] 29 Phone: +34 954557179; fax: +34 954556447. 30 1. Introduction 31 Plastic materials are currently considered very important materials due to their 32 exceptional properties and performance over other materials such as metal and wood 33 (Plastic Europe, 2008). In fact, according to a recent report, the demand for plastic will 34 continue to rise following a trend that has increased since 1950s (Plastics Europe et 35 al., 2008). Nowadays, the substitution of petroleum-based plastics with bio-based 36 plastics is seen as a promising alternative because it will reduce the dependency of 37 plastics on fossil fuels and the pressure on landfills from plastic solid wastes (Alvarez- 38 Chavez et al., 2012). In recent years there has been a great interest to utilize 39 renewable biomass in the manufacture of high-quality, cost-competitive and 40 biodegradable consumer goods as a means to reduce the consumption and the 41 dependence on petrochemical feedstock and to diminish environmental pollution 42 (Rosentrater and Otieno, 2006; Tummala et al., 2006). In particular, packaging films 43 and containers made of natural biopolymers represent a particular interest due to their 44 biodegradability, since most of these products have a relative short service life ending 45 up in landfills. In this sense, protein-based materials have been proved to be 46 completely degrading in 50 days when buried in farmland soils (Domenek et al., 2004). 47 Proteins, lipids and polysaccharides have been proposed as biopolymers sources 48 for many years (Averous, 2004; De Graaf, 2000; Hernández-Izquierdo and Krochta, 49 2008; Irissin-Mangata et al., 2001; Siracusa et al., 2008). Regarding proteins, most 50 studies have used plant proteins such as zein, wheat gluten or soybean to manufacture 51 bioplastics (Cuq et al., 1998; Gomez-Martinez et al., 2013; Jerez et al., 2005; Kim, 52 2008; Zheng et al., 2003). Moreover, some works have been focused on animal 53 3 proteins such as milk proteins, collagen, gelatin, etc. (Cuq et al., 1998; Pommet et al., 54 2003). 55 Soy protein is the major coproduct of soybean oil and is one of the cheapest 56 proteins in nature (Tian et al., 2012). In fact, soy proteins have commonly been used 57 for food and animal feed for many years. However, soy protein is a new polymer for 58 biodegradable resins. Soy protein polymers are macromolecules that contain a number 59 of amino acids and side chains that can be used in the manufacture of plastic (Sun et 60 al., 1999). The mechanical properties of soy protein based plastics can be controlled 61 and optimized by adjusting the initial moisture content as well as some processing 62 parameters such as the molding temperature and/or pressure (Wang et al., 2007; Liang 63 et al., 1999; Mo et al., 1999; Jane and Wang, 1996; Pateau et al., 1994). However, the 64 application of soy protein plastics is limited because of its low strength (Tummala et al., 65 2006) and high moisture absorption (Liu et al., 2005). Therefore, it can be concluded 66 that the most effective method is to blend soy protein with another biodegradable 67 polymer. Currently, soy-based blends for plastic applications include polyphosphate 68 (Otaigbe and Adams, 1997), polyesters (Graiver et al., 2004; Liu et al., 2004), 69 polyurethane (Tian et al., 2010) or natural fibers (Liu et al., 2005). On the other hand, 70 no reports on the enhancement of soy-based plastic materials by using a combination 71 with other proteins have been found. 72 Egg white protein (albumen), traditionally used by the food industry, has recently 73 proved the feasibility to produce bioplastics (Jerez et al., 2007b). Moreover, if 74 compared to other common proteins like gluten, egg white has proved to be an 75 adequate raw material in the obtaining of highly-transparent bioplastics with suitable 76 mechanical properties for the manufacture of biodegradable food packaging and other 77 plastic products. Blends of this protein with other products from agricultural sources, 78 biodegradable and of lower cost than protein and synthetic additives have been 79 recently proposed (González-Gutierrez et al., 2010). 80 4 A protein-based material could be defined as a stable three-dimensional 81 macromolecular network stabilized and strengthened by hydrogen bonds, hydrophobic 82 interactions and disulfide bonds (Pommet et al., 2003). However, as proteins 83 themselves do not have sufficient plasticity to be handled and brittle properties are 84 typically found in bioplastics, a plasticizer is required. The role of plasticizers in 85 reducing the glass transition temperature and providing mobility to polymeric chains 86 has been extensively described (Irissin-Mangata et al., 2001; Matveev et al., 2000; 87 Pouplin et al., 1999). Due to its excellent properties, glycerol is among the most 88 commonly used plasticizers for biopolymer-based biodegradable materials. 89 Protein/plasticizer blends (bioplastics) can be processed using existing plastic 90 processing technologies: from the physic-chemical or casting method (Gennadios, 91 2002) to thermo-plastic/mechanical method (compression molding or extrusion) (Jerez 92 et al., 2007a; González-Gutiérrez et al., 2011). However, a relevant technique such as 93 injection molding, which is among the most common processing methods used with 94 synthetic polymers, has not been found to be used for protein-based bioplastic 95 applications. The potential use of protein-based injection molding to produce many 96 kinds of shaped products will entail new arguments in favor of considering these 97 biopolymer materials as an alternative to synthetic plastics for different applications. 98 The overall objective has been to study plasticized albumen/soy biobased plastic 99 materials processed by injection molding. To achieve this objective, different 100 albumen/soy ratios plasticized with glycerol have been processed. In addition, some 101 variables such as temperature and residence time in the pre-injection mixing chamber, 102 as well as the temperature of the mold have been also analyzed in order to select 103 suitable processing parameters for protein-based injection molding. 104 2. Material and methods 105 2.1. Materials 106 5 Commercial spray-dried albumen (AP) was provided by OVOSEC S.A and soy 107 protein isolate (SPI) was supplied by Protein Technologies International (SUPRO 108 500E, Leper, Belgium). The protein content of both products was determined in 109 quadruplicate as % N x 6.25 using a LECO CHNS-932 nitrogen micro analyzer (Leco 110 Corporation, St. Joseph, MI, USA) (Etheridge et al., 1998) being 83 wt.% for AP and 91 111 wt.% for SPI. Glycerol (GL), from Panreac Química, S.A. (Spain), was used as protein 112 plasticizer. 113 2.2. Sample preparation 114 Blends containing 60 wt.% protein, with different AP/SPI ratios as shown in Table 1, 115 and 40 wt.% glycerol (GL) were mixed in a two-blade counter-rotating batch mixer 116 (Brabender Plastograph, Germany). Mixing process was carried out at 25ºC and 50 117 rpm for c.a. 10 min (Jerez et al., 2005) to obtain a dough-like material at neutral pH. 118 The final pH value was measured by a Crison pH 25 pHmeter in combination with a 119 puncture electrode (Crison Instruments S.A., Barcelona, Spain). 120 The dough-like materials obtained after the mixing process were subsequently 121 processed by injection molding using a MiniJet Piston Injection Molding System 122 (ThermoHaake, Karlsruhe, Germany) to obtain bioplastic probes. The most suitable 123 processing variables, such as injection temperature and pressure, as well as residence 124 time in the pre-injection mixing chamber, were selected after performing temperature 125 ramp and time sweep tests to the dough-like materials. Two types of molds were used 126 to prepare the probes: a 60×10×1 mm rectangular shape mold for both DMTA 127 experiments and transparency measurements and a Dumpbell type probe defined by 128 ISO 527-2:1993 for Tensile Properties of Plastics. 129 2.3. Characterization 130 2.3.1. Protein solubility. Protein solubility at different pH values was determined. 131 Aqueous dispersions (c.a. 1.00 g protein/40 mL) were prepared and pH of different 132 aliquots was adjusted to alkaline pH values with 6 N NaOH, and to acid pH with 2 N 133 6 HCl. Samples were homogenized and subsequently centrifuged for 20 min at 10,000 x 134 g at 10ºC. The supernatant were collected for protein content determination by means 135 of the Markwell method (Markwell et al., 1978). Solubility was expressed as a 136 percentage (g soluble protein/100 g isolate in sample). 137 2.3.2. Protein extractability. Samples were extracted in different extraction media (2.5 138 mL): distilled water; a denaturing agent solution (Method A); a solution of denaturing 139 and reducing agents (Method B). All the materials were extracted for 2h at 20ºC by 140 magnetic agitation (approximately 400 rpm). Method A used a 0.086 mol L-1 Tris-base, 141 0.045 mmol·L-1 glycine, 2 mmol·L-1 EDTA, 10 g·L-1, 5 g·L-1 Sodium Dodecyl Sulfate 142 (SDS) pH 8 buffer. In method B, proteins were dissolved in the same buffer containing 143 10 g·L-1 Dithiothreitol (DTT). Dispersions were centrifuged at 10,000 x g for 15 min at 144 15ºC and protein content was determined by a LECO CHNS-932 nitrogen micro 145 analyzer. Similar methods were previously used by the authors with protein gels 146 (Romero et al., 2011). Protein solubility was determined from supernatant and 147 expressed as 100x protein content in the supernatant/total protein content. Three 148 independent extractions were carried out with each solvent. Average values (±standard 149 deviation) are reported. 150 2.3.3. Free and total sulfhydryls. Free and total sulfhydryl groups of protein samples 151 were determined using the method developed by Beveridge et al. (1974) and 152 Thannhauser et al. (1984), respectively. Samples were suspended (1 mg/mL) in 0.086 153 mol/L Tris-HCl – 0.09 mol/L glycine – 4 mmol/L EDTA – 8 mol/L urea – pH 8 buffer. 154 Dispersions were stirred at 25 ºC during 10 min at 500 rpm in a thermomixer and then 155 centrifuged at 15,000g (10 min, 10 ºC). Supernatant was incubated with Ellman’s 156 reagent (4mg DTNB/mL methanol) and 1 mL NTSB was used in the case of the total 157 sulfhydryls. Absorbance at 412 nm was measured in a Genesis-20 spectrophotometer 158 (Thermo Scientific, USA). The molar extinction coefficient of NTB (13,600 L·mol-1·cm-1) 159 7 was used. Protein concentration of extracts was determined by the Bradford method 160 (Bradford, 1976). 161 2.3.4. Surface hydrophobicity of proteins (H0). Surface hydrophobicity (H0) of soluble 162 proteins from protein extracts (pH 8) was measured according to Kato and Nakai 163 (1980), using the fluorescent probe 1-anilino-8-naphtalene-sulfonate (ANS). Protein 164 extract were diluted with pH 8-0.05 M phosphate buffer to obtain protein concentrations 165 ranging from 5 to 0.005 mg/mL. Then, 40 μL of ANS (8.0 mM in the same buffer) were 166 added to 2 mL of sample. Fluorescence intensity (FI) was measured with a Perkin- 167 Elmer 2000 fluorescence spectrometer (Perkin-Elmer Corp. Norwalk, CT, USA), at 168 wavelengths of 365 nm (excitation) and 484 nm (emission). The initial slope of 169 fluorescence intensity versus protein concentration plot was used as an index of 170 protein hydrophobicity (H0). 171 2.3.5. Differential Scanning Calorimetry (DSC). DSC experiments were performed with 172 a Q20 (TA Instruments, USA), using 5 to 10 mg samples, in hermetic aluminum pans. 173 A heating rate of 10 ºC/min was selected. The sample was purged with a nitrogen flow 174 of 50 mL/min. 175 2.3.6. Rheological measurements. Dough-like material was characterized by Small 176 Amplitude Oscillatory Shear (SAOS) measurements, using a controlled-strain 177 rheometer (ARES), in order to select the optimum conditions for injection molding. The 178 geometry used has been a plate and plate geometry (dia: 25 mm) with a rough surface 179 and a gap between plates of 1 mm. Low viscosity Dow Corning 200 fluid has been 180 used as sealant to avoid sample drying. Strain sweep SAOS tests were also performed 181 in order to establish the linear viscoelasticity range. Temperature ramp tests were 182 carried out at 5 ºC/min from 20 to 100 ºC and time sweep tests were performed for 183 1800 s at a selected constant temperature. In these measurements, complex viscosity 184 (  *) was monitored at a constant frequency of 2 rad/s. All the systems studied had the 185 same thermorheological history before performing any rheological test. 186 8 2.3.7. Dynamic Mechanical Temperature Analysis (DMTA). DMTA tests were carried 187 out with a RSA3 (TA Instruments, New Castle, DE, USA), on rectangular probes using 188 dual cantilever bending. All the experiments were carried out at constant frequency 189 (1Hz) and strain (between 0.01 and 0.3%, within the linear viscoelastic region). The 190 selected heating rate was 3ºC min−1. All the samples were coated with Dow Corning 191 high vacuum grease to avoid water loss. 192 2.3.8. Tensile strength measurements. Tensile tests were performed by using the 193 Insight 10 kN Electromechanical Testing System (MTS, Eden Prairie, MN, USA), 194 according to by ISO 527-2:1993 for Tensile Properties of Plastics. Tensile stress and 195 elongation at break were evaluated from at least three duplicates for each product 196 using type IV probes and an extensional rate of 100 mm·min−1 at room temperature. 197 2.3.9. Water absorption capacity. Water uptake of bioplastics was determined following 198 the ASTM D570 norm (ASTM D570-98, Standard test Method for Water Absorption of 199 Plastics) (ASTM, 2001) using at least three 60×10×1 mm specimens immersed in 200 distillate water for 2h or 24 h at room temperature. 201 2.3.10. Transparency Measurements. Transparency measurements were done by 202 means of a Genesis-20 spectrophotometer (Thermo Scientific, USA). Transmittance 203 (%) of rectangular specimens, 3 mm thick, was measured using a wavelength of 600 204 nm. Air was used as blank (100% transmittance). A transmittance index (IT) was 205 defined in order to compare the transparency of the different bioplastic. 206 𝐼𝑇=𝐵𝑖𝑜𝑝𝑙𝑎𝑠𝑡𝑖𝑐 𝑇𝑟𝑎𝑛𝑠𝑚𝑖𝑡𝑡𝑎𝑛𝑐𝑒 (%) 𝐴𝑙𝑏𝑢𝑚𝑖𝑛 − 𝑏𝑎𝑠𝑒𝑑 𝐵𝑖𝑜𝑝𝑙𝑎𝑠𝑡𝑖𝑐 𝑇𝑟𝑎𝑛𝑠𝑚𝑖𝑡𝑡𝑎𝑛𝑐𝑒 (%) ∙100 (1) 207 2.4. Statistical analysis 208 At least three replicates of each measurement were carried out. Statistical analyses 209 were performed using t-test and one-way analysis of variance (ANOVA, p0.05) by 210 9 means of the statistical package SPSS 18. Standard deviations from some selected 211 parameters were calculated. 212 3. Results and discussion 213 3.1. Protein solubility 214 Fig. 1 shows the solubility-pH profile obtained for both AP and SPI systems. AP 215 system displays high solubility values within the whole experimental range of pH 216 values, showing values higher than 80 wt.%. These results support the high hydrophilic 217 character of AP system which is clearly dominated by its high content in highly soluble 218 albumin proteins. Thus, almost 70% of protein in egg white is ovoalbumin and 219 conalbumin (Cheftel et al., 1989). This high solubility takes place even at the minimum 220 shown in Fig.1 observed at pH 5.8-6, which should correspond to the isoelectric point 221 (IEP). However, the IEP for egg white has been reported to be slightly lower since Loeb 222 (1923) reported a value of 4.8, while the IEP is located at 5.4 according to Riddick 223 (1968). These values are consistent with the results obtained from z-potential 224 measurements (data not shown) performed for this AP system that exhibited a null 225 value at pH around 5. 226 SPI exhibits much lower solubility values than AP system, showing a maximum 227 solubility (ca. 36%) at pH 8 and a minimum (ca. 7.2%) at pH 6. However, a wide low- 228 solubility range (lower than 10%) may be noticed between pH 3 and 6. Molina-Ortiz et 229 al. (2004) reported higher solubility values, particularly at acidic pH, with a much 230 narrower minimum at pH 4.5 for SPI prepared from a non thermally treated soy flour (to 231 avoid protein denaturation). Thus, the extension of the low solubility profile to the 3-6 232 pH range might be attributed to some protein denaturation during SPI manufacture. 233 It is also interesting to mention that the solubility profile shows a second minimum at 234 pH 9. This minimum, although being less apparent, has been also detected by Franzen 235 and Kinsella (1976) for a SPI showing much higher solubility at alkaline pH. 236 3.2. Disulfide and sulfhydryl groups 237 16 protein-based bioplastic materials (Jerez et al., 2007a, 2007b; Zárate-Ramírez et al., 401 2011), indicates that some thermosetting potential still remains after injection molding 402 at the experimental conditions. These results confirm that the conditions selected for 403 injection molding, summarized in Table 5, are more suitable for SPI-based probes. As 404 for the AP/SPI-based bioplastics, the behavior is much closer to that one found for the 405 SPI/GL system. The AP/SPI mixed systems show an intermediate behavior, being only 406 apparent at temperatures higher than 60ºC. 407 The higher viscoelastic properties shown by the system containing AP as the only 408 protein may be related to some of the physicochemical properties previously shown. 409 Thus, the lowest temperature of the DSC peak shown in Fig. 3 (much lower than the 410 processing temperature) would lead to a high degree of denaturation for this system. 411 This effect would lead to a higher surface hydrophobocity that in any case is higher for 412 AP (Table 3) that would produce a higher increase in protein aggregation. In addition, 413 the higher value of free sulfhydryls shown by AP (Table 2) would also yield a higher 414 density of crosslinking by forming hydrogen bonds. Similar results have been reported 415 by Bounocore et al. (2003) that also explained this behavior in terms of thermal 416 induced crosslinking. 417 All the probes studied display similar loss tangent profiles (Fig. 4B) showing one 418 single peak, which is related to a glass-like transition of the plasticized protein-based 419 material, with tan 𝛿 values ranging from 0.1 to 0.5, excepting for the AP/GL system that 420 shows a much lower tan 𝛿 peak at ca. 0.34. These unimodal profiles indicate a good 421 compatibility between protein and glycerol, obtained for all the systems after the 422 injection molding process, regardless of the protein used (AP, SPI or AP/SPI mixtures). 423 The value of the temperature at the peak for the AP/GL system is ca. 63ºC, which is 424 rather coincident with those values previously found for albumen-based bioplastic 425 prepared by compression molding with the same protein/glycerol ratio (Jerez et al., 426 2007a, González-Gutierrez et al., 2010). As may be observed in the plot included in 427 Fig.4B, this temperature at the peak increases asymptotically with the relative 428 17 proportion of SPI in the protein mixture up to a value of ca. 78.5ºC. This earlier 429 transition found for AP/GL bioplastics is also consistent with the earlier Tg obtained for 430 AP/GL blends from DSC measurements. 431 3.8. Uniaxial tensile strength measurements 432 The results obtained from uniaxial strength measurements are shown in Fig. 5. Fig. 433 5A displays the results of stress-strain curves obtained for protein-based bioplastics 434 using AP, SPI and 50:50 AP/SPI mixtures. 435 All the curves exhibit an initial linear elastic behavior of high constant stress-strain 436 slope yielding high values for the Young’s Modulus (E), followed by a plastic 437 deformation stage with a continuous decrease in the stress-strain slope after the elastic 438 limit. A second constant slope is reached at the end of this plastic deformation stage. 439 All the curves eventually reach a maximum value for the stress (max) and the strain 440 (εmax), which is immediately followed by a sudden decrease in stress that corresponds 441 to the rupture of the sample. Fig. 5B shows the values of the three parameters (E, max 442 and εmax) from tensile tests performed on AP/SPI/GL bioplastic probes, as a function of 443 the albumen content in the AP/SPI mixture. Parameters (max) and E show a 444 progressive increase with increasing albumen content, with an overall growth in the 445 order of 50% for both parameters. Parameter εmax also undergoes an increase up to 446 50% AP concentration. However, a further increase up to 100% does not lead to any 447 significant difference. Therefore, it may be generally stated that AP-based bioplastics 448 exhibit better tensile properties than SPI-based systems in accordance to the highest 449 values also found for the elastic modulus from DMA measurements and the lowest Td 450 obtained with DSC tests. 451 In any case, all the Protein/GL bioplastic probes exhibit tensile properties lower than 452 synthetic polymers such as LDPE. Thus, parameters max, εmax and E show values that 453 reach as much as 15%, 18% and 10%, respectively, of the values for ASTM 454 normalized LDPE (ASTM D638). 455 18 3.9. Water uptake capacity 456 Fig. 6 shows the results from water uptake measurements obtained after immersion 457 of bioplastic samples for 2 and 24 h, as well as the water-soluble matter loss, as a 458 function of AP content. 459 No significant differences between water uptake percentages after 2 and 24 h are 460 found, regardless of the AP/SPI ratio used in the bioplastic preparation by injection 461 molding. This fact indicates occurrence of relatively faster water absorption kinetics as 462 compared to gluten-based bioplastics processed by compression molding (unpublished 463 results). In addition, an increase in AP content induces a progressive decrease in water 464 absorption up to half the value corresponding to the AP free sample. It may be pointed 465 out that this last value is much lower than those previously reported (Jerez et al., 466 2007a) for similar AP/GL samples. However, these samples used higher AP/GL ratio (2 467 instead of 1.5) and were prepared by compression molding. Although the former fact 468 may induce some changes, the above-mentioned difference in water uptake capacity 469 may be mainly attributed to the different thermomechanical processing conditions used. 470 In fact, as reported by Jerez et al. (2007a), an increase in pressure led to a remarkable 471 reduction in water absorption. 472 On the other hand, the fact that AP-based bioplastic display lower absorption 473 capacity than SPI-based samples may be related to a higher structure degree of the 474 AP protein matrix. This is consistent with the above-mentioned higher protein 475 crosslinking obtained for AP/GL bioplastic probes as deduced from DMA and tensile 476 tests. This behavior is in agreement with those results found by other authors 477 (Buonocore et al., 2003; Zheng et al., 2003), who reported that the swelling ratio of 478 polymer matrices decrease with increasing protein crosslinking. 479 As regards the water-soluble loss matter the AP/GL system shows a value of 40% 480 and all the SPI-containing probes exhibit values that are slightly higher. These results 481 suggest that the loss of soluble matter corresponds basically to the highly hydrophilic 482 glycerol. In fact, all the samples contain 40% GL. The extra-loss matter observed for 483 19 SPI-based samples should correspond to moisture content as well as to some protein 484 that is not strongly associated to the network structure. 485 3.10. Transparency measurements 486 Fig. 7 shows the evolution of the transmittance index (IT) with increasing AP 487 percentage in the total protein content of the injection molded bioplastic probe. Images 488 of each probe are also included in Fig. 7. The evolution of color and transparency with 489 increasing AP content is apparent, leading to a remarkable increase in the value of IT. 490 The value obtained for AP-based probe is 46.8  0.4, being slightly lower than the 491 value corresponding to LDPE (54.0 ± 0.3) and 52% higher than that of the SPI-based 492 probe. Moreover, as may be noted, addition of 25% AP causes the highest increase in 493 IT value representing about 63% of the total increase in transmittance. 494 4. Concluding remarks 495 AP concentrate shows higher solubility than SPI. AP also shows higher sulfhydryl 496 groups and disulfide bonds, as well as higher hydrophobicity. Therefore, AP 497 concentrate evidences higher potentials for thermomechanical processing than SPI. 498 According to the extractability analysis of protein concentrates and protein/GL 499 blends, some shear-induce effects took place during the mixing process although being 500 less noticeable for AP than for SPI systems. As a result of the mixing process, glycerol 501 reduced the glass transition and denaturation temperatures to a higher extent when 502 SPI protein was used instead of AP. The combination of temperature ramps and time 503 sweep tests at selected temperature has demonstrated to be suitable techniques in 504 order to select operation conditions for injection molding. These processing conditions 505 took into account the mixed thermoplastic and thermoset character of the protein 506 systems studied where the contribution of the later is more relevant. 507 Both protein systems used alone or in combination yield bioplastics that can be 508 thermomechanically processed by injection molding, although showing lower 509 20 rheological and mechanical properties than LDPE standards, under the selected 510 processing conditions. 511 A comparison between AP and SPI-based injection molded bioplastic specimens 512 revealed that the former show higher viscoelastic bending properties and higher 513 uniaxial tensile properties. However, under the selected conditions, SPI still showed a 514 remaining thermosetting potential. In any case, as a consequence of the lower 515 rheological and mechanical properties of the later, SPI showed a higher water uptake 516 capacity. 517 The results of transmittance put forward that injection molding may yield highly 518 transparent protein-based polymeric materials by adding a moderate proportion of AP 519 to the raw protein/plasticiser blend. Some of these materials showed transparency 520 properties that were even comparable to LDPE plastic materials. These results are 521 consistent with those reported by other previous researchers that used egg white- 522 based compression molded materials. 523 Acknowledgements 524 This work is part of a research project sponsored by Andalousian Government, 525 (Spain) (project TEP-6134) and by “Ministerio de Economía y Competitividad” from 526 Spanish Government (Ref. MAT2011-29275-C02-02/01). The authors gratefully 527 acknowledge their financial support. 528 References 529 ASTM, 2001. 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