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Biothermoplastics from soyproteins by steaming
Industrial Crops and Products 36 (2012) 116–121
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Biothermoplastics from soyproteins by steaming Yiqi Yang a,b,c , Narendra Reddy a,∗ a b c
Department of Textiles, Clothing & Design, 234, HECO Building, East Campus, University of Nebraska-Lincoln, Lincoln, NE 68583-0802, United States Department of Biological Systems Engineering, 234, HECO Building, East Campus, University of Nebraska-Lincoln, Lincoln, NE 68583-0802, United States Nebraska Center for Materials and Nanoscience, 234, HECO Building, East Campus, University of Nebraska-Lincoln, Lincoln, NE 68583-0802, United States
a r t i c l e
i n f o
Article history: Received 12 April 2011 Received in revised form 29 July 2011 Accepted 13 August 2011 Available online 24 September 2011 Keywords: Soyproteins Thermoplastic Tensile strength Steaming Moisture
a b s t r a c t We report a novel method of developing thermoplastics from steamed soyproteins with good tensile properties. Soyproteins are generally made thermoplastic by using plasticizers or by chemical modifications. However, soyprotein thermoplastics developed using plasticizers have poor tensile properties when wet and chemical modifications make soyproteins expensive and/or environmentally unfriendly. In this research, soyproteins were steamed at various temperatures and time and the steamed proteins were compression molded into thermoplastic films. The effect of steaming on the molecular weight and thermal behavior and tensile properties of the films at different steaming and compression conditions were studied. Steaming substantially reduced the molecular weights, decreased the melting temperature and increased the melting enthalpy. Thermoplastics developed from steamed soyproteins had good tensile strength (5 MPa) and modulus (193 MPa) but moderate elongation (14.5%). Although glycerol was necessary to improve the thermoplasticity, soyprotein thermoplastics developed in this research required lower glycerol to form thermoplastic films compared to films reported in literature. Steaming of soyproteins shows promise to be an inexpensive and environmentally friendly process to develop biothermoplastics. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Thermoplastics have been developed from soy and other plant proteins for food, packaging and other industrial applications (Wang et al., 2008; Otaigbe et al., 1999; Swain et al., 2004). Plant protein based thermoplastics are biodegradable, derived from renewable resources and are therefore preferable over synthetic polymer based thermoplastics (Zhang and Mittal, 2010; Guerrero and Caba, 2010; Sun et al., 2007, 2008). However, plant proteins are inherently non-thermoplastic and it is necessary to use plasticizers or chemically modify the proteins to make them thermoplastic. Soyproteins have been mixed with various ratios (30–50%) of glycerol and used to develop thermoplastic films by compression molding (Guerrero and Caba, 2010). It was found that compression molded films have high tensile strength and elongation than solution cast films. The tensile strength and modulus decreased whereas elongation increased with increasing the amount of glycerol in the films (Guerrero and Caba, 2010). Soyprotein isolates were mixed with glycerol and compression molded at 150 ◦ C for 2 min at a pressure of 10 MPa to form thermoplastic films and the viscoelastic, thermal and microstructural properties were studied (Ogale et al., 2000). In addition to glyc-
∗ Corresponding author. Tel.: +1 402 472 3020; fax: +1 402 472 0640. E-mail address: [email protected] (N. Reddy). 0926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2011.08.018
erol, other plasticizers such as ethylene glycol and propylene glycol have also been used to develop thermoplastics from soyproteins (Swain et al., 2004; Wu and Zhang, 2001a,b). Although plasticizers made soyproteins thermoplastic and improved the flexibility of the thermoplastics, using plasticizers substantially decreased the tensile strength especially under high humidities or in water (Wu and Zhang, 2001a,b). Similar effect has also been observed for thermo-molded films developed from sunflower proteins (Orliac et al., 2003). Attempts have also been made to blend soyproteins with other natural and synthetic polymers to improve the mechanical properties and water stability. Soyproteins were blended with starch and the pressure–volume–temperature relationships on the thermal behavior and specific volume were studied (Otaigbe and Jane, 1997). It was found that blending poly(lactic acid) (PLA) with soyproteins increased the tensile strength, elongation and water resistance of the thermoplastics (Zhang et al., 2006). However, the tensile strength and elongation of the PLA blended soyprotein thermoplastics were considerably lower compared to soyprotein thermoplastics developed by other researchers. Various concentrations of cellulose whiskers were mixed with soyprotein isolates and 30% glycerol was used to compression mold at 140 ◦ C for 10 min at a pressure of 20 MPa. Adding cellulose whiskers (30%) increased the tensile strength from 5.8 to 8.1 MPa and Young’s modulus from 44.7 to 133.2 MPa compared to pure soyprotein isolates (Wang et al., 2006). In another report, soypro-
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teins were acetylated and compression molded to form films without the need for plasticizers. Films with tensile strength ranging from 1.8 to 2.5 MPa and elongation ranging from 73% to 113% were obtained depending on the temperature of processing and thickness of the films (Foulk and Bunn, 2001). However, the films had low wet tensile strength ranging from 0.4 to 0.8 MPa and wet elongation ranging from 16% to 34% (Foulk and Bunn, 2001). It has been well documented that heat, pressure, compression and/or pH alter the physiochemical properties of proteins. The effect of pH on the thermal and mechanical properties of glycerol plasticized soyprotein films has been recently studied (Guerrero and Caba, 2010; Guerrero et al., 2010). It was found that tensile strength and elongation were higher at basic pH’s due to better unfolding of the proteins (Guerrero and Caba, 2010; Guerrero et al., 2010). High pressure is reported to affect protein conformation leading to protein denaturation, aggregation or gelation. Therefore, the influence of high pressure on the functional properties (protein solubility, emulsifying activity index and rheological properties) of soyproteins dispersed in solution were investigated (Torezzan et al., 2007; Puppo et al., 2004). It was found that high pressure caused molecular unfolding, decrease in thermal stability and an increase in protein solubility. Similar to pressure, steaming of soyproteins at 100 ◦ C for various times was found to decrease protein solubility and trypsin inhibitor activity (Anderson, 1992). Steaming was reported to cleave disulfide bonds, reduce the molecular weight and decompose high sulfur containing protein fractions in wool (Tonin et al., 2006). Except for pH, the influences of other pretreatment conditions on the thermoplasticity and mechanical properties of thermoplastics developed from soyproteins have not been studied. In addition, in all of the approaches discussed above, the thermoplastics developed have poor mechanical properties, especially when wet. This is true even for acetylated soyproteins (Foulk and Bunn, 2001). It is necessary to have soyprotein thermoplastics with good dry and wet mechanical properties to be able to use the thermoplastics for industrial applications. So far, it has not been possible to obtain protein thermoplastics with good dry and wet tensile properties and thermoplasticity required to develop products. In this research, we have studied the effect of steaming on the thermal behavior and tensile properties of compression molded soyprotein thermoplastic films both in the dry and wet conditions.
2. Materials and methods 2.1. Materials Soyprotein isolates (PRO-FAM® 646 isolated soy protein with 90% protein) were supplied by Archer Daniels Midlands Company, Decatur, IL. Glycerol was purchased from VWR international.
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2.3. Thermal analysis The untreated and soyproteins steamed at various temperatures were analyzed in a differential scanning calorimeter to determine the thermoplasticity. About 8–10 mg of the samples were dried at 50 ◦ C for 12 h to remove moisture and then placed in sealed aluminum pans and heated in a Mettler Toledo DSC (Model 822e) from 25 to 200 ◦ C at a heating rate of 20 ◦ C/min. Scans obtained were analyzed to determine the melting temperature and melting enthalpy. 2.4. Electrical resistance We determined the electrical resistance of the soyproteins steamed at different conditions at various compression temperatures and time. Soyprotein powder was spread on a piece of fabric and one end of the two probes from the conductivity meter were placed in the soyprotein at a fixed distance apart ensuring that the probes were completely covered with the proteins. The other two ends of the probe were connected to the conductivity meter. A piece of fabric was placed on top and the assembly was placed between the two hot plates of the compression mold. The mold was gradually heated and the electrical resistance was recorded with increasing temperature at a fixed compressive pressure of 30,000 PSI. The resistance decreased with increasing temperature and the soyproteins were considered to melt when the resistance was zero. The temperature of zero resistance was used to compression mold the soyproteins into thermoplastics. 2.5. SDS-PAGE Changes in the molecular weight of the soyproteins due to steaming were studied using SDS-PAGE. Samples were dispersed in distilled water (1 mg/ml) and heated at 70 ◦ C for approximately 10 min. One ml of the protein dispersion was collected and 8 l of reducing agent (NUPAGE, Invitrogen) and 20 l of 4× running buffer (NUPAGE LDS, Invitrogen) was added into the dispersion. About 20 l of the dispersion was loaded into NUPAGE–Bis–Tris Pre-cast gels. The gel was run at 200 V for 40 min and standard molecular weight ladders (Invitrogen) were used for identification. 2.6. Developing thermoplastics Soyprotein powder was thoroughly mixed by hand with 15 or 30% (w/w) glycerol and the mixture was evenly spread on aluminum sheets. The soyprotein-glycerol mixture was placed between two hot plates and compression molded at various temperatures (120–200 ◦ C) for different periods of time (2–8 min) at 280 MPa. After compression, the hot press was cooled by running cold water and the films formed were collected for further analysis. At least three samples were compression molded separately for each steaming condition. 2.7. Morphology
2.2. Steaming soyproteins The soyprotein isolate powder was placed inside high pressure steel canisters in paper bags. A fixed amount of distilled water was poured inside the canisters and the canisters were tightly sealed and heated at various temperatures (110–140 ◦ C) and time (1–4 h) in a hot air oven for the steaming to occur. It was ensured that the soyproteins were not in contact with water in the canister and the same weight of soyproteins and water was used for steaming each time. After steaming, the canisters were cooled by running cold water and the steamed soyproteins were collected.
Thermoplastic films obtained from soyproteins using the conditions mentioned above were observed under a scanning electron microscope (SEM) (Hitachi S3000N) to study the surface morphology. Samples were fixed on conductive adhesive tapes and sputter coated with gold palladium before observing in the microscope at a voltage of 25 kV. 2.8. Tensile properties Films were conditioned in a standard testing atmosphere of 21 ◦ C and 65% relative humidity for at least 24 h before testing.
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Fig. 2. SDS-PAGE of soyproteins before and after steaming at 120, 130 and 140 ◦ C without reduction (lanes 1–4) and with reduction (lanes 5–8) during electrophoresis.
Fig. 1. SDS-PAGE of soyproteins before (lane 2) and after steaming at 120 (lane 3), 130 (lane 4) and 140 ◦ C (lane 5) for 2 h. Lane 1 is the molecular weight standard.
Samples measuring (8 cm × 1.5 cm) were cut from the films and tested for their tensile properties according to ASTM standard D882. Gauge length was 2 inches and crosshead speed was 10 mm/min. At least six samples each from three different films were tested for each steaming or compression condition. Data obtained was analyzed to determine the average and ±one standard deviation. 2.9. Wet tensile properties The thermoplastic soyprotein films were dipped in distilled water at 21 ◦ C for 30 min. Samples were taken out and tested for the wet tensile properties according to ASTM standard D882 as described above. 3. Results and discussion 3.1. Effect of steaming on the molecular weight of the proteins SDS image in Fig. 1 shows that steaming considerably decreased the molecular weight of the proteins. Proteins steamed at 120 ◦ C (lane 3) have some high molecular weight proteins in the region of 36–40 kDa but many proteins originally with molecular weights of 36–40 kDa in lane 2 have lower molecular weights and are predominantly seen in the region of 10–14 kDa in lane 3. Increasing steaming temperature to 130 ◦ C further reduces the molecular weighs and there are very few proteins in the 36–40 kDa region
as seen from lane 4. Most of the proteins have molecular weights less than 14 kDa. Proteins steamed at 140 ◦ C (lane 5) have been considerably degraded and there are no proteins remaining in the 36–40 kDa region. Most of the proteins have low molecular weights, lower than 15 kDa. Steaming hydrolyses and also degrades the proteins leading to a decrease in molecular weight (Anderson, 1992; Tonin et al., 2006). It has been reported that steam cleaves disulfide bonds and makes proteins in wool to have lower molecular weights and melting temperature (Tonin et al., 2006). Similar phenomenon was observed for the soyproteins steamed at different conditions. However, steaming did not completely hydrolyze the disulfide bonds. Fig. 2 shows the SDS-PAGE image of soyproteins with and without reduction of disulfide bonds during electrophoresis. Lane 1 in Fig. 2 shows the unsteamed soyprotein and lanes 2, 3 and 4 are soyproteins steamed at 120, 130 and 140 ◦ C, respectively without reduction during electrophoresis. Lanes 5–8 are the proteins corresponding to the proteins in lanes 1–4 after being reduced during electrophoresis. The reduced proteins show more prominent and distinct bands than the corresponding non- reduced proteins. For instance, lane 7 has more distinct and clearer bands than lane 3 suggesting that steaming did not completely break the disulfide bonds. 3.2. Effect of steaming on electrical resistance during compression molding The electrical resistance of the soyproteins steamed at different conditions was measured to determine the most optimum steaming condition. Fig. 3 shows the changes in the electrical resistance of soyproteins steamed at different temperatures and compression molded at 150 ◦ C and 280 PSI. As seen from the figure, steaming
Y. Yang, N. Reddy / Industrial Crops and Products 36 (2012) 116–121
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Table 1 Effect of steaming temperature on the properties of thermoplastic soyprotein films compressed at 150 ◦ C for 2 min.
Fig. 3. Electrical resistance of soyproteins after steaming at different temperatures and times and compression molded at 150 ◦ C.
at high temperatures decreased the electrical resistance of the soyproteins. Proteins steamed at 100 ◦ C for 3 and 4 h had much higher resistance than the proteins steamed at higher temperatures indicating that steaming at 100 ◦ C was not effective in denaturing the proteins and making them thermoplastic. Soyproteins steamed at 120 ◦ C for 1 h had similar electrical resistance as soyproteins steamed at 110 ◦ C for 2 h. Increasing the steaming time to 2 h at 120 ◦ C substantially decreases the electrical resistance. Steaming at 130 ◦ C just for 1 h provided soyproteins with low electrical resistance. The electrical resistance of the soyproteins steamed at 130 ◦ C for 2 h was about 6 k and therefore the soyproteins were steamed at 130 ◦ C for 2 h to develop thermoplastics. Although higher steaming temperatures may have decreased the steaming time further, it would not be convenient to handle steaming above 130 ◦ C and most of the proteins were denatured when steamed at 140 ◦ C for 2 h as seen from Fig. 1. 3.3. Effect of steaming on thermal behavior of soyproteins Changes in the melting behavior of soyproteins after steaming at different conditions are shown in Fig. 4. Steaming makes the soyproteins thermoplastic as indicated by the decrease in melting temperature and increase in melting enthalpy. The unsteamed soyproteins show a small melting peak with an melting enthalpy of 78 J/g at about 154 ◦ C. Steaming at 110 ◦ C for 2 h did not change the melting temperature but increased the melting enthalpy by about 50% suggesting that the proteins became more thermoplas-
Fig. 4. Melting temperature and melting enthalpy of soyproteins steamed at different temperatures obtained by heating the soyproteins in a DSC at a rate of 20 ◦ C/min.
Steaming temperature (◦ C)
Tensile strength (MPa)
Breaking elongation (%)
Young’s modulus (MPa)
120 130 140
2.4 ± 0.9 5.0 ± 0.8 2.7 ± 0.7
5.6 ± 1.4 14.5 ± 3.0 5.9 ± 1.4
210 ± 40 193 ± 60 183 ± 57
tic. Further increase in steaming temperature to 130 ◦ C decreased the melting temperature to 143 ◦ C and substantially increased the melting enthalpy to 274 J/g. However, higher steaming temperature (140 ◦ C) decreased the melting temperature further to 122 ◦ C but decreased the melting enthalpy to 164 J/g. Steaming at high temperature (140 ◦ C) hydrolyzed and denatured the proteins leading to lower molecular weight proteins as seen by the SDS-PAGE in Fig. 1. Proteins with lower molecular weights melt at low temperatures leading to a broad melting peak with a lower melting temperature and melting enthalpy compared to steaming at 130 ◦ C. The DSC also indicated that steaming at 130 ◦ C for 2 h was the most optimum to obtain thermoplastic soyproteins with good thermoplasticity and lesser damage to the proteins. 3.4. Tensile properties of thermoplastics developed from steamed soyproteins 3.4.1. Effect of steaming temperature Table 1 shows the effect of steaming temperature on the tensile properties of the soyprotein thermoplastic films. Although proteins steamed at 110 ◦ C showed a melting peak, the proteins did not melt and the films were non-homogenous with unmelted soyproteins. Therefore, thermoplastics developed from soyproteins steamed at 110 ◦ C were not tested for tensile properties. Thermoplastic soyprotein films with the highest tensile strength and breaking elongation were obtained with soyproteins steamed at 130 ◦ C for 2 h. Increasing steaming temperature considerably decreased the tensile strength and elongation of the films due to the damage to the proteins as indicated by SDS-PAGE. Soyproteins steamed at 130 ◦ C had good thermoplasticity and formed homogenous films as seen from the SEM image in Fig. 5. The comparatively high elongation of the films obtained after steaming at 130 ◦ C also suggests that the proteins had melted and made the films flexible. A previous study has reported that molecular weight of soyproteins affects the tensile properties of soyprotein films and higher molecular
Fig. 5. SEM image of the thermoplastic soyprotein film shows that the proteins have melted and there are very few aggregates.
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Table 2 Effect of compression temperature on the properties of thermoplastic soyprotein films. Soyprotein was steamed at 130 ◦ C for 2 h and compression molded for 2 min. Compression temperature (◦ C)
Tensile strength (MPa)
120 150 180 200
2.2 5.0 2.8 1.7
± ± ± ±
0.6 0.8 0.3 0.2
Breaking elongation (%) 3.4 14.5 7.7 5.7
± ± ± ±
Table 3 Effect of compression time on the properties of thermoplastic soyprotein films. Soyprotein was steamed at 130 ◦ C for 2 h and compression molded for 150 ◦ C.
Young’s modulus (MPa)
1.4 3.0 2.4 2.5
210 193 183 147
± ± ± ±
40 60 57 53
weights provide better tensile properties (Wu and Zhang, 2001a,b). Although proteins with higher molecular weights provide better tensile properties, proteins with lower molecular weights have better thermoplasticity and could form more homogenous films with better tensile properties (Table 2). 3.4.2. Effect of compression temperature Similar to steaming temperature, compression temperature also influenced the tensile properties of the films. Compressing at 120 ◦ C resulted in films with low tensile strength and breaking elongation but with high modulus mainly due to the incomplete melting of the soyproteins. The temperature and time was not sufficient to melt the soyprotein and produce homogenous films with good flexibility. Increasing temperature to 150 ◦ C more than doubled the tensile strength and increased the elongation 4 times compared to the properties of the films obtained at 120 ◦ C. However, further increase in compression temperature decreased all the tensile properties due to the damage to the soyproteins. Films made at 200 ◦ C had turned brown indicating thermal degradation of the proteins. Other researchers have also reported that a compression temperature of 150 ◦ C was most optimum to process soyproteins plasticized with glycerol (Ogale et al., 2000; Guerrero and Caba, 2010; Guerrero et al., 2010; Mo et al., 1999). 3.4.3. Effect of compression time Increasing compression time above 2 min decreased the peak stress and elongation as seen from Table 3 when compression molded at 150 ◦ C. The strength, elongation and modulus of the films decreased considerably when compression molded for 8 min. As with temperature, prolonged exposure to heat damages the proteins and decreases the tensile properties. However, using temperatures lower than 150 ◦ C and time longer than 2 min may provide similar properties as those obtained at 150 ◦ C and 2 min.
Compression time (Min)
Tensile strength (MPa)
2 4 6 8
5.0 4.2 4.5 3.3
± ± ± ±
0.8 0.7 0.8 0.9
Breaking elongation (%) 14.5 10.0 10.2 8.5
± ± ± ±
3.0 3.1 2.2 2.6
Young’s modulus (MPa) 193 190 187 160
± ± ± ±
60 37 30 47
3.4.4. Effect of glycerol and water on tensile properties Effect of increasing glycerol on the tensile properties of the soyprotein thermoplastics is shown in Table 4. With 30% glycerol, the tensile strength of the films decreased by about 54%, modulus decreased by 72% whereas elongation was relatively unaffected. The elongation of the films obtained in this research is lower than the elongation of films obtained by other researchers at similar concentrations of glycerol and compression conditions. Differences in the proteins used and conditions (temperature or humidity) of testing most likely contributed to these differences. Poor wet tensile properties are a major limitation of thermoplastics developed from soyproteins and other biopolymers, especially when plasticizers are used (Foulk and Bunn, 2001). Although the wet strength and modulus of the films obtained in this research are much lower than those in the dry state, the wet strength and modulus of the films are much higher than those of previously reported soyprotein thermoplastic films. 3.5. Comparison of tensile properties of soyprotein thermoplastics with literature data Table 5 provides a comparison of the tensile properties of thermoplastic soyprotein films prepared by various researchers with the properties of films obtained in this research. As seen from the table, the thermoplastic films developed in this research had better tensile strength and modulus but lower elongation than most of the soyprotein films reported in literature. Films were obtained in this research using 15% glycerol whereas all the previous reports have used 25–50% glycerol as the plasticizer and found that increasing the content of plasticizer increases the elongation but considerably decreased the strength and modulus. As seen from the table, films made from 50% glycerol have substantially lower tensile strength and modulus than those with lower amounts of glycerol. For comparison, we also used 30% glycerol and found that
Table 4 Effect of concentration of glycerol on the dry and wet tensile properties of the soyprotein films. Soyprotein was steamed at 130 ◦ C for 2 h and compression molded for 150 ◦ C for 2 min. Glycerol (% w/w)
15 30 a
Tensile strength (MPa) a
Breaking elongation (%)
Young’s modulus (MPa)
Dry
Wet
Dry
Wet
Dry
Wet
5.0 ± 0.8 2.3 ± 0.4
0.6 ± 0.1 0.5 ± 0.1
14.5 ± 3.0 16.5 ± 5.2
8.7 ± 2.7 15.4 ± 3.5
193 ± 60 55 ± 6
11 ± 1.5 4.5 ± 1
In water at 21 ◦ C for 30 min.
Table 5 Comparison of the properties of soyprotein thermoplastics reported in literature to the films developed in this research. Plasticizer (%w/w) or chemical modification
Tensile strength (MPa)
Breaking elongation (%)
Young’s modulus (MPa)
Reference
15% Glycerol 30% Glycerol 50% Glycerol 30–50% Glycerol 30% Glycerol 25% Glycerol Acetylated soyproteins
5.0 ± 0.8 2.3 ± 0.4 3–7 2.8–7.8 5–7 35–45 1.8–2.5
14.5 ± 3.0 16.5 ± 5.2 60–135 132–186 20–150 5–7 73–113
193 ± 60 55 ± 6 NA 15–120 NA 1000–1100 NA
This paper This paper Wu and Zhang (2001a,b) Guerrero and Caba (2010) Guerrero and Caba (2010) Mo et al. (1999) Foulk and Bunn (2001)
Y. Yang, N. Reddy / Industrial Crops and Products 36 (2012) 116–121
increasing glycerol reduced the tensile strength by about 55% and the modulus by about 70% but there was no significant change in the breaking elongation. However, it should be noted that the film forming and testing conditions considerably affect the tensile properties and the data in the table were obtained at varying compression and testing conditions. 4. Conclusions This research shows that thermoplastics developed from steamed soyproteins have better dry and wet tensile properties compared to most thermoplastic soyprotein films previously developed from plasticized or chemically modified soyproteins. Steaming cleaves disulfide bonds, hydrolyses and denatures the proteins leading to a substantial decrease in the molecular weight. However, steaming increased the thermoplasticity as indicated by the lower melting temperatures and higher melting enthalpies. Compression molding conditions affected the tensile properties. Steaming at 130 ◦ C for 2 h and compression molding at 150 ◦ C for 2 min was found to provide an optimum tensile strength of 5 MPa, breaking elongation of 14.5% and modulus of 193 MPa. The tensile strength and modulus of the thermoplastic films obtained in this research were higher than most of the soyprotein films reported in literature. However, the breaking elongation of the thermoplastics obtained in this research was lower than previous reports at similar concentrations of glycerol. The thermoplastic films developed in this research were stable in water. Steaming of soyproteins shows potential to provide useful thermoplastics from soyproteins. Acknowledgements We thank the Agricultural Research Division at the University of Nebraska-Lincoln, Multi State Project S-1026 and USDA Hatch act for the financial support to complete this research. References Anderson, R.L., 1992. Effects of steaming on soybean proteins and trypsin inhibitors. J. Am. Oil Chem. Soc. 69 (12), 1170–1176. Foulk, J.A., Bunn, J.M., 2001. Properties of compression molded acetylated soy protein films. Ind. Crop Prod. 14, 11–22.
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Biothermoplastics from soyproteins by steaming Yiqi Yang a,b,c , Narendra Reddy a,∗ a b c
Department of Textiles, Clothing & Design, 234, HECO Building, East Campus, University of Nebraska-Lincoln, Lincoln, NE 68583-0802, United States Department of Biological Systems Engineering, 234, HECO Building, East Campus, University of Nebraska-Lincoln, Lincoln, NE 68583-0802, United States Nebraska Center for Materials and Nanoscience, 234, HECO Building, East Campus, University of Nebraska-Lincoln, Lincoln, NE 68583-0802, United States
a r t i c l e
i n f o
Article history: Received 12 April 2011 Received in revised form 29 July 2011 Accepted 13 August 2011 Available online 24 September 2011 Keywords: Soyproteins Thermoplastic Tensile strength Steaming Moisture
a b s t r a c t We report a novel method of developing thermoplastics from steamed soyproteins with good tensile properties. Soyproteins are generally made thermoplastic by using plasticizers or by chemical modifications. However, soyprotein thermoplastics developed using plasticizers have poor tensile properties when wet and chemical modifications make soyproteins expensive and/or environmentally unfriendly. In this research, soyproteins were steamed at various temperatures and time and the steamed proteins were compression molded into thermoplastic films. The effect of steaming on the molecular weight and thermal behavior and tensile properties of the films at different steaming and compression conditions were studied. Steaming substantially reduced the molecular weights, decreased the melting temperature and increased the melting enthalpy. Thermoplastics developed from steamed soyproteins had good tensile strength (5 MPa) and modulus (193 MPa) but moderate elongation (14.5%). Although glycerol was necessary to improve the thermoplasticity, soyprotein thermoplastics developed in this research required lower glycerol to form thermoplastic films compared to films reported in literature. Steaming of soyproteins shows promise to be an inexpensive and environmentally friendly process to develop biothermoplastics. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Thermoplastics have been developed from soy and other plant proteins for food, packaging and other industrial applications (Wang et al., 2008; Otaigbe et al., 1999; Swain et al., 2004). Plant protein based thermoplastics are biodegradable, derived from renewable resources and are therefore preferable over synthetic polymer based thermoplastics (Zhang and Mittal, 2010; Guerrero and Caba, 2010; Sun et al., 2007, 2008). However, plant proteins are inherently non-thermoplastic and it is necessary to use plasticizers or chemically modify the proteins to make them thermoplastic. Soyproteins have been mixed with various ratios (30–50%) of glycerol and used to develop thermoplastic films by compression molding (Guerrero and Caba, 2010). It was found that compression molded films have high tensile strength and elongation than solution cast films. The tensile strength and modulus decreased whereas elongation increased with increasing the amount of glycerol in the films (Guerrero and Caba, 2010). Soyprotein isolates were mixed with glycerol and compression molded at 150 ◦ C for 2 min at a pressure of 10 MPa to form thermoplastic films and the viscoelastic, thermal and microstructural properties were studied (Ogale et al., 2000). In addition to glyc-
∗ Corresponding author. Tel.: +1 402 472 3020; fax: +1 402 472 0640. E-mail address: [email protected] (N. Reddy). 0926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2011.08.018
erol, other plasticizers such as ethylene glycol and propylene glycol have also been used to develop thermoplastics from soyproteins (Swain et al., 2004; Wu and Zhang, 2001a,b). Although plasticizers made soyproteins thermoplastic and improved the flexibility of the thermoplastics, using plasticizers substantially decreased the tensile strength especially under high humidities or in water (Wu and Zhang, 2001a,b). Similar effect has also been observed for thermo-molded films developed from sunflower proteins (Orliac et al., 2003). Attempts have also been made to blend soyproteins with other natural and synthetic polymers to improve the mechanical properties and water stability. Soyproteins were blended with starch and the pressure–volume–temperature relationships on the thermal behavior and specific volume were studied (Otaigbe and Jane, 1997). It was found that blending poly(lactic acid) (PLA) with soyproteins increased the tensile strength, elongation and water resistance of the thermoplastics (Zhang et al., 2006). However, the tensile strength and elongation of the PLA blended soyprotein thermoplastics were considerably lower compared to soyprotein thermoplastics developed by other researchers. Various concentrations of cellulose whiskers were mixed with soyprotein isolates and 30% glycerol was used to compression mold at 140 ◦ C for 10 min at a pressure of 20 MPa. Adding cellulose whiskers (30%) increased the tensile strength from 5.8 to 8.1 MPa and Young’s modulus from 44.7 to 133.2 MPa compared to pure soyprotein isolates (Wang et al., 2006). In another report, soypro-
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teins were acetylated and compression molded to form films without the need for plasticizers. Films with tensile strength ranging from 1.8 to 2.5 MPa and elongation ranging from 73% to 113% were obtained depending on the temperature of processing and thickness of the films (Foulk and Bunn, 2001). However, the films had low wet tensile strength ranging from 0.4 to 0.8 MPa and wet elongation ranging from 16% to 34% (Foulk and Bunn, 2001). It has been well documented that heat, pressure, compression and/or pH alter the physiochemical properties of proteins. The effect of pH on the thermal and mechanical properties of glycerol plasticized soyprotein films has been recently studied (Guerrero and Caba, 2010; Guerrero et al., 2010). It was found that tensile strength and elongation were higher at basic pH’s due to better unfolding of the proteins (Guerrero and Caba, 2010; Guerrero et al., 2010). High pressure is reported to affect protein conformation leading to protein denaturation, aggregation or gelation. Therefore, the influence of high pressure on the functional properties (protein solubility, emulsifying activity index and rheological properties) of soyproteins dispersed in solution were investigated (Torezzan et al., 2007; Puppo et al., 2004). It was found that high pressure caused molecular unfolding, decrease in thermal stability and an increase in protein solubility. Similar to pressure, steaming of soyproteins at 100 ◦ C for various times was found to decrease protein solubility and trypsin inhibitor activity (Anderson, 1992). Steaming was reported to cleave disulfide bonds, reduce the molecular weight and decompose high sulfur containing protein fractions in wool (Tonin et al., 2006). Except for pH, the influences of other pretreatment conditions on the thermoplasticity and mechanical properties of thermoplastics developed from soyproteins have not been studied. In addition, in all of the approaches discussed above, the thermoplastics developed have poor mechanical properties, especially when wet. This is true even for acetylated soyproteins (Foulk and Bunn, 2001). It is necessary to have soyprotein thermoplastics with good dry and wet mechanical properties to be able to use the thermoplastics for industrial applications. So far, it has not been possible to obtain protein thermoplastics with good dry and wet tensile properties and thermoplasticity required to develop products. In this research, we have studied the effect of steaming on the thermal behavior and tensile properties of compression molded soyprotein thermoplastic films both in the dry and wet conditions.
2. Materials and methods 2.1. Materials Soyprotein isolates (PRO-FAM® 646 isolated soy protein with 90% protein) were supplied by Archer Daniels Midlands Company, Decatur, IL. Glycerol was purchased from VWR international.
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2.3. Thermal analysis The untreated and soyproteins steamed at various temperatures were analyzed in a differential scanning calorimeter to determine the thermoplasticity. About 8–10 mg of the samples were dried at 50 ◦ C for 12 h to remove moisture and then placed in sealed aluminum pans and heated in a Mettler Toledo DSC (Model 822e) from 25 to 200 ◦ C at a heating rate of 20 ◦ C/min. Scans obtained were analyzed to determine the melting temperature and melting enthalpy. 2.4. Electrical resistance We determined the electrical resistance of the soyproteins steamed at different conditions at various compression temperatures and time. Soyprotein powder was spread on a piece of fabric and one end of the two probes from the conductivity meter were placed in the soyprotein at a fixed distance apart ensuring that the probes were completely covered with the proteins. The other two ends of the probe were connected to the conductivity meter. A piece of fabric was placed on top and the assembly was placed between the two hot plates of the compression mold. The mold was gradually heated and the electrical resistance was recorded with increasing temperature at a fixed compressive pressure of 30,000 PSI. The resistance decreased with increasing temperature and the soyproteins were considered to melt when the resistance was zero. The temperature of zero resistance was used to compression mold the soyproteins into thermoplastics. 2.5. SDS-PAGE Changes in the molecular weight of the soyproteins due to steaming were studied using SDS-PAGE. Samples were dispersed in distilled water (1 mg/ml) and heated at 70 ◦ C for approximately 10 min. One ml of the protein dispersion was collected and 8 l of reducing agent (NUPAGE, Invitrogen) and 20 l of 4× running buffer (NUPAGE LDS, Invitrogen) was added into the dispersion. About 20 l of the dispersion was loaded into NUPAGE–Bis–Tris Pre-cast gels. The gel was run at 200 V for 40 min and standard molecular weight ladders (Invitrogen) were used for identification. 2.6. Developing thermoplastics Soyprotein powder was thoroughly mixed by hand with 15 or 30% (w/w) glycerol and the mixture was evenly spread on aluminum sheets. The soyprotein-glycerol mixture was placed between two hot plates and compression molded at various temperatures (120–200 ◦ C) for different periods of time (2–8 min) at 280 MPa. After compression, the hot press was cooled by running cold water and the films formed were collected for further analysis. At least three samples were compression molded separately for each steaming condition. 2.7. Morphology
2.2. Steaming soyproteins The soyprotein isolate powder was placed inside high pressure steel canisters in paper bags. A fixed amount of distilled water was poured inside the canisters and the canisters were tightly sealed and heated at various temperatures (110–140 ◦ C) and time (1–4 h) in a hot air oven for the steaming to occur. It was ensured that the soyproteins were not in contact with water in the canister and the same weight of soyproteins and water was used for steaming each time. After steaming, the canisters were cooled by running cold water and the steamed soyproteins were collected.
Thermoplastic films obtained from soyproteins using the conditions mentioned above were observed under a scanning electron microscope (SEM) (Hitachi S3000N) to study the surface morphology. Samples were fixed on conductive adhesive tapes and sputter coated with gold palladium before observing in the microscope at a voltage of 25 kV. 2.8. Tensile properties Films were conditioned in a standard testing atmosphere of 21 ◦ C and 65% relative humidity for at least 24 h before testing.
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Fig. 2. SDS-PAGE of soyproteins before and after steaming at 120, 130 and 140 ◦ C without reduction (lanes 1–4) and with reduction (lanes 5–8) during electrophoresis.
Fig. 1. SDS-PAGE of soyproteins before (lane 2) and after steaming at 120 (lane 3), 130 (lane 4) and 140 ◦ C (lane 5) for 2 h. Lane 1 is the molecular weight standard.
Samples measuring (8 cm × 1.5 cm) were cut from the films and tested for their tensile properties according to ASTM standard D882. Gauge length was 2 inches and crosshead speed was 10 mm/min. At least six samples each from three different films were tested for each steaming or compression condition. Data obtained was analyzed to determine the average and ±one standard deviation. 2.9. Wet tensile properties The thermoplastic soyprotein films were dipped in distilled water at 21 ◦ C for 30 min. Samples were taken out and tested for the wet tensile properties according to ASTM standard D882 as described above. 3. Results and discussion 3.1. Effect of steaming on the molecular weight of the proteins SDS image in Fig. 1 shows that steaming considerably decreased the molecular weight of the proteins. Proteins steamed at 120 ◦ C (lane 3) have some high molecular weight proteins in the region of 36–40 kDa but many proteins originally with molecular weights of 36–40 kDa in lane 2 have lower molecular weights and are predominantly seen in the region of 10–14 kDa in lane 3. Increasing steaming temperature to 130 ◦ C further reduces the molecular weighs and there are very few proteins in the 36–40 kDa region
as seen from lane 4. Most of the proteins have molecular weights less than 14 kDa. Proteins steamed at 140 ◦ C (lane 5) have been considerably degraded and there are no proteins remaining in the 36–40 kDa region. Most of the proteins have low molecular weights, lower than 15 kDa. Steaming hydrolyses and also degrades the proteins leading to a decrease in molecular weight (Anderson, 1992; Tonin et al., 2006). It has been reported that steam cleaves disulfide bonds and makes proteins in wool to have lower molecular weights and melting temperature (Tonin et al., 2006). Similar phenomenon was observed for the soyproteins steamed at different conditions. However, steaming did not completely hydrolyze the disulfide bonds. Fig. 2 shows the SDS-PAGE image of soyproteins with and without reduction of disulfide bonds during electrophoresis. Lane 1 in Fig. 2 shows the unsteamed soyprotein and lanes 2, 3 and 4 are soyproteins steamed at 120, 130 and 140 ◦ C, respectively without reduction during electrophoresis. Lanes 5–8 are the proteins corresponding to the proteins in lanes 1–4 after being reduced during electrophoresis. The reduced proteins show more prominent and distinct bands than the corresponding non- reduced proteins. For instance, lane 7 has more distinct and clearer bands than lane 3 suggesting that steaming did not completely break the disulfide bonds. 3.2. Effect of steaming on electrical resistance during compression molding The electrical resistance of the soyproteins steamed at different conditions was measured to determine the most optimum steaming condition. Fig. 3 shows the changes in the electrical resistance of soyproteins steamed at different temperatures and compression molded at 150 ◦ C and 280 PSI. As seen from the figure, steaming
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Table 1 Effect of steaming temperature on the properties of thermoplastic soyprotein films compressed at 150 ◦ C for 2 min.
Fig. 3. Electrical resistance of soyproteins after steaming at different temperatures and times and compression molded at 150 ◦ C.
at high temperatures decreased the electrical resistance of the soyproteins. Proteins steamed at 100 ◦ C for 3 and 4 h had much higher resistance than the proteins steamed at higher temperatures indicating that steaming at 100 ◦ C was not effective in denaturing the proteins and making them thermoplastic. Soyproteins steamed at 120 ◦ C for 1 h had similar electrical resistance as soyproteins steamed at 110 ◦ C for 2 h. Increasing the steaming time to 2 h at 120 ◦ C substantially decreases the electrical resistance. Steaming at 130 ◦ C just for 1 h provided soyproteins with low electrical resistance. The electrical resistance of the soyproteins steamed at 130 ◦ C for 2 h was about 6 k and therefore the soyproteins were steamed at 130 ◦ C for 2 h to develop thermoplastics. Although higher steaming temperatures may have decreased the steaming time further, it would not be convenient to handle steaming above 130 ◦ C and most of the proteins were denatured when steamed at 140 ◦ C for 2 h as seen from Fig. 1. 3.3. Effect of steaming on thermal behavior of soyproteins Changes in the melting behavior of soyproteins after steaming at different conditions are shown in Fig. 4. Steaming makes the soyproteins thermoplastic as indicated by the decrease in melting temperature and increase in melting enthalpy. The unsteamed soyproteins show a small melting peak with an melting enthalpy of 78 J/g at about 154 ◦ C. Steaming at 110 ◦ C for 2 h did not change the melting temperature but increased the melting enthalpy by about 50% suggesting that the proteins became more thermoplas-
Fig. 4. Melting temperature and melting enthalpy of soyproteins steamed at different temperatures obtained by heating the soyproteins in a DSC at a rate of 20 ◦ C/min.
Steaming temperature (◦ C)
Tensile strength (MPa)
Breaking elongation (%)
Young’s modulus (MPa)
120 130 140
2.4 ± 0.9 5.0 ± 0.8 2.7 ± 0.7
5.6 ± 1.4 14.5 ± 3.0 5.9 ± 1.4
210 ± 40 193 ± 60 183 ± 57
tic. Further increase in steaming temperature to 130 ◦ C decreased the melting temperature to 143 ◦ C and substantially increased the melting enthalpy to 274 J/g. However, higher steaming temperature (140 ◦ C) decreased the melting temperature further to 122 ◦ C but decreased the melting enthalpy to 164 J/g. Steaming at high temperature (140 ◦ C) hydrolyzed and denatured the proteins leading to lower molecular weight proteins as seen by the SDS-PAGE in Fig. 1. Proteins with lower molecular weights melt at low temperatures leading to a broad melting peak with a lower melting temperature and melting enthalpy compared to steaming at 130 ◦ C. The DSC also indicated that steaming at 130 ◦ C for 2 h was the most optimum to obtain thermoplastic soyproteins with good thermoplasticity and lesser damage to the proteins. 3.4. Tensile properties of thermoplastics developed from steamed soyproteins 3.4.1. Effect of steaming temperature Table 1 shows the effect of steaming temperature on the tensile properties of the soyprotein thermoplastic films. Although proteins steamed at 110 ◦ C showed a melting peak, the proteins did not melt and the films were non-homogenous with unmelted soyproteins. Therefore, thermoplastics developed from soyproteins steamed at 110 ◦ C were not tested for tensile properties. Thermoplastic soyprotein films with the highest tensile strength and breaking elongation were obtained with soyproteins steamed at 130 ◦ C for 2 h. Increasing steaming temperature considerably decreased the tensile strength and elongation of the films due to the damage to the proteins as indicated by SDS-PAGE. Soyproteins steamed at 130 ◦ C had good thermoplasticity and formed homogenous films as seen from the SEM image in Fig. 5. The comparatively high elongation of the films obtained after steaming at 130 ◦ C also suggests that the proteins had melted and made the films flexible. A previous study has reported that molecular weight of soyproteins affects the tensile properties of soyprotein films and higher molecular
Fig. 5. SEM image of the thermoplastic soyprotein film shows that the proteins have melted and there are very few aggregates.
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Table 2 Effect of compression temperature on the properties of thermoplastic soyprotein films. Soyprotein was steamed at 130 ◦ C for 2 h and compression molded for 2 min. Compression temperature (◦ C)
Tensile strength (MPa)
120 150 180 200
2.2 5.0 2.8 1.7
± ± ± ±
0.6 0.8 0.3 0.2
Breaking elongation (%) 3.4 14.5 7.7 5.7
± ± ± ±
Table 3 Effect of compression time on the properties of thermoplastic soyprotein films. Soyprotein was steamed at 130 ◦ C for 2 h and compression molded for 150 ◦ C.
Young’s modulus (MPa)
1.4 3.0 2.4 2.5
210 193 183 147
± ± ± ±
40 60 57 53
weights provide better tensile properties (Wu and Zhang, 2001a,b). Although proteins with higher molecular weights provide better tensile properties, proteins with lower molecular weights have better thermoplasticity and could form more homogenous films with better tensile properties (Table 2). 3.4.2. Effect of compression temperature Similar to steaming temperature, compression temperature also influenced the tensile properties of the films. Compressing at 120 ◦ C resulted in films with low tensile strength and breaking elongation but with high modulus mainly due to the incomplete melting of the soyproteins. The temperature and time was not sufficient to melt the soyprotein and produce homogenous films with good flexibility. Increasing temperature to 150 ◦ C more than doubled the tensile strength and increased the elongation 4 times compared to the properties of the films obtained at 120 ◦ C. However, further increase in compression temperature decreased all the tensile properties due to the damage to the soyproteins. Films made at 200 ◦ C had turned brown indicating thermal degradation of the proteins. Other researchers have also reported that a compression temperature of 150 ◦ C was most optimum to process soyproteins plasticized with glycerol (Ogale et al., 2000; Guerrero and Caba, 2010; Guerrero et al., 2010; Mo et al., 1999). 3.4.3. Effect of compression time Increasing compression time above 2 min decreased the peak stress and elongation as seen from Table 3 when compression molded at 150 ◦ C. The strength, elongation and modulus of the films decreased considerably when compression molded for 8 min. As with temperature, prolonged exposure to heat damages the proteins and decreases the tensile properties. However, using temperatures lower than 150 ◦ C and time longer than 2 min may provide similar properties as those obtained at 150 ◦ C and 2 min.
Compression time (Min)
Tensile strength (MPa)
2 4 6 8
5.0 4.2 4.5 3.3
± ± ± ±
0.8 0.7 0.8 0.9
Breaking elongation (%) 14.5 10.0 10.2 8.5
± ± ± ±
3.0 3.1 2.2 2.6
Young’s modulus (MPa) 193 190 187 160
± ± ± ±
60 37 30 47
3.4.4. Effect of glycerol and water on tensile properties Effect of increasing glycerol on the tensile properties of the soyprotein thermoplastics is shown in Table 4. With 30% glycerol, the tensile strength of the films decreased by about 54%, modulus decreased by 72% whereas elongation was relatively unaffected. The elongation of the films obtained in this research is lower than the elongation of films obtained by other researchers at similar concentrations of glycerol and compression conditions. Differences in the proteins used and conditions (temperature or humidity) of testing most likely contributed to these differences. Poor wet tensile properties are a major limitation of thermoplastics developed from soyproteins and other biopolymers, especially when plasticizers are used (Foulk and Bunn, 2001). Although the wet strength and modulus of the films obtained in this research are much lower than those in the dry state, the wet strength and modulus of the films are much higher than those of previously reported soyprotein thermoplastic films. 3.5. Comparison of tensile properties of soyprotein thermoplastics with literature data Table 5 provides a comparison of the tensile properties of thermoplastic soyprotein films prepared by various researchers with the properties of films obtained in this research. As seen from the table, the thermoplastic films developed in this research had better tensile strength and modulus but lower elongation than most of the soyprotein films reported in literature. Films were obtained in this research using 15% glycerol whereas all the previous reports have used 25–50% glycerol as the plasticizer and found that increasing the content of plasticizer increases the elongation but considerably decreased the strength and modulus. As seen from the table, films made from 50% glycerol have substantially lower tensile strength and modulus than those with lower amounts of glycerol. For comparison, we also used 30% glycerol and found that
Table 4 Effect of concentration of glycerol on the dry and wet tensile properties of the soyprotein films. Soyprotein was steamed at 130 ◦ C for 2 h and compression molded for 150 ◦ C for 2 min. Glycerol (% w/w)
15 30 a
Tensile strength (MPa) a
Breaking elongation (%)
Young’s modulus (MPa)
Dry
Wet
Dry
Wet
Dry
Wet
5.0 ± 0.8 2.3 ± 0.4
0.6 ± 0.1 0.5 ± 0.1
14.5 ± 3.0 16.5 ± 5.2
8.7 ± 2.7 15.4 ± 3.5
193 ± 60 55 ± 6
11 ± 1.5 4.5 ± 1
In water at 21 ◦ C for 30 min.
Table 5 Comparison of the properties of soyprotein thermoplastics reported in literature to the films developed in this research. Plasticizer (%w/w) or chemical modification
Tensile strength (MPa)
Breaking elongation (%)
Young’s modulus (MPa)
Reference
15% Glycerol 30% Glycerol 50% Glycerol 30–50% Glycerol 30% Glycerol 25% Glycerol Acetylated soyproteins
5.0 ± 0.8 2.3 ± 0.4 3–7 2.8–7.8 5–7 35–45 1.8–2.5
14.5 ± 3.0 16.5 ± 5.2 60–135 132–186 20–150 5–7 73–113
193 ± 60 55 ± 6 NA 15–120 NA 1000–1100 NA
This paper This paper Wu and Zhang (2001a,b) Guerrero and Caba (2010) Guerrero and Caba (2010) Mo et al. (1999) Foulk and Bunn (2001)
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increasing glycerol reduced the tensile strength by about 55% and the modulus by about 70% but there was no significant change in the breaking elongation. However, it should be noted that the film forming and testing conditions considerably affect the tensile properties and the data in the table were obtained at varying compression and testing conditions. 4. Conclusions This research shows that thermoplastics developed from steamed soyproteins have better dry and wet tensile properties compared to most thermoplastic soyprotein films previously developed from plasticized or chemically modified soyproteins. Steaming cleaves disulfide bonds, hydrolyses and denatures the proteins leading to a substantial decrease in the molecular weight. However, steaming increased the thermoplasticity as indicated by the lower melting temperatures and higher melting enthalpies. Compression molding conditions affected the tensile properties. Steaming at 130 ◦ C for 2 h and compression molding at 150 ◦ C for 2 min was found to provide an optimum tensile strength of 5 MPa, breaking elongation of 14.5% and modulus of 193 MPa. The tensile strength and modulus of the thermoplastic films obtained in this research were higher than most of the soyprotein films reported in literature. However, the breaking elongation of the thermoplastics obtained in this research was lower than previous reports at similar concentrations of glycerol. The thermoplastic films developed in this research were stable in water. Steaming of soyproteins shows potential to provide useful thermoplastics from soyproteins. Acknowledgements We thank the Agricultural Research Division at the University of Nebraska-Lincoln, Multi State Project S-1026 and USDA Hatch act for the financial support to complete this research. References Anderson, R.L., 1992. Effects of steaming on soybean proteins and trypsin inhibitors. J. Am. Oil Chem. Soc. 69 (12), 1170–1176. Foulk, J.A., Bunn, J.M., 2001. Properties of compression molded acetylated soy protein films. Ind. Crop Prod. 14, 11–22.
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