Rheological Studies, Production, and Characterization of Injection

Injection-Molded Plastics from Sunflower Protein Isolate ... The rheological properties of a sunflower protein isolate (SFPI) were studied, and inject...
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Ind. Eng. Chem. Res. 2003, 42, 1674-1680

MATERIALS AND INTERFACES Rheological Studies, Production, and Characterization of Injection-Molded Plastics from Sunflower Protein Isolate Olivier Orliac,* Franc¸ oise Silvestre, Antoine Rouilly, and Luc Rigal Laboratoire de Chimie Agro-industrielle, UMR 1010 INRA/INP-ENSIACET, 118, route de Narbonne, F-31077 Toulouse Cedex 04, France

The rheological properties of a sunflower protein isolate (SFPI) were studied, and injectionmolded pieces were produced. Various mixtures of SFPI (100 parts by weight), glycerol (10-50 parts), water (10-50 parts), and sometimes sodium sulfite (used as reducing agent, 1-10 parts) were studied. Power-law-type modeling of the viscosity as a function of the moisture and glycerol contents described the thermoplastic properties of the proteins under certain conditions. The presence of sodium sulfite in the mixture seemed to improve the flow properties substantially, with an optimum at 4 parts (w/w). Injection-molded objects were then made, and their mechanical properties were tested. The maximum tensile strength (16.1 MPa) was obtained for 4 parts of glycerol (with 100 and 18 parts of protein and water, respectively) and decreased with increasing glycerol content to 10.6 MPa for 22 parts of glycerol. The elastic modulus was between 2.0 and 0.5 GPa, with a strain at break of no more than 1.8%. 1. Introduction Sunflower seed storage proteins are a renewable and abundant source of raw materials for the production of plastics. These proteins are extracted from sunflower oil cake, which is an industrial byproduct resulting from the extraction of oil. They are biodegradable and can be used to make environmentally friendly materials. In this era, when environmental constraints are becoming increasingly stricter , plastics derived from agricultural materials are a potentially valuable alternative to traditional plastics derived from petrochemical products. Several studies have been carried out in our laboratory to valorize sunflower oil cake proteins. These studies have been shaped by studies on cast films1 and on thermo-molded films.2,3 This study is the continuation of these earlier studies and concerns the molding and the properties of injectable materials made from sunflower protein isolate. Injection was chosen because it is widely used for thermoplastic materials. This molding process consists of two phases: First, the material is softened by thermal and mechanical treatment involving a screw extrusion system. Second, the material is injected through a pipe into a closed mold. Few studies have addressed the injection of proteins or, more generally, biopolymers. At the end of the 1970s, Shirai et al.4 developed an injection procedure for a mixture of wheat gluten and soy flour for the manufacture of products for food use. However, it was not until 10 years later that a procedure was developed that exploited the hydration properties of proteins as a means of controlling the viscosity and therefore made it possible to adjust the molding condi* To whom correspondence should be addressed. E-mail: [email protected]. Tel.: +33-562-885725. Fax: +33562-885730.

tions. Wittwer and Tomka have also used gelatin to make capsules by injection.5 They similarly made injected pieces from starch, which had been shown in the 1960s to be susceptible to plasticization by water.6,7 Subsequently, a few studies have been conducted on the injection of zein-starch mixtures,8 soy proteinstarch mixtures,9 and soy proteins.10,11 The main goals of this study were (i) to study the flow properties of sunflower proteins as a function of their thermal properties, (ii) to optimize the amounts of plasticizers and additives present in the mixtures in terms of their rheological properties for injection, and (iii) to make injection-molded objects and study their mechanical properties. 2. Experimental Section 2.1. Materials and Methods. The sunflower protein extract was obtained by alkaline extraction from sunflower oil cake on a semi-industrial scale (composition shown in Table 1). After centrifugation, the soluble proteins are precipitated at their isoelectric point by the addition of concentrated sulfuric acid. A second centrifugation then allows their separation from the aqueous phase. The third stage consists of spray drying at 50 °C, followed by conditioning in 15-kg bags. The glycerol used as a plasticizer and the sodium sulfite used as a reducing agent were “reagent” grade and were purchased from Aldrich (St. Quentin Fallavier, France). These agents were used without prior treatment. The sunflower protein isolate, glycerol, and water, and sodium sulfite as appropriate, were mixed using a Perrier 32.00 mixer (Montrouge, France). The mixture generally contained 100 parts (by weight) of sunflower protein isolate (SFPI), 10-50 parts of glycerol, and 10-

10.1021/ie020913x CCC: $25.00 © 2003 American Chemical Society Published on Web 03/19/2003

Ind. Eng. Chem. Res., Vol. 42, No. 8, 2003 1675 Table 1. Characterization of Sunflower Oil Cake and Isolate of Sunflower Proteins (SFPIa) sunflower oil cakeb

SFPIb

determination method

10 ( 1 7.6 ( 0.4 34.4 ( 1.0 1.0 ( 0.5 22.3 ( 2.0 5.2 ( 1.0 9.8 ( 1.0 5.7 ( 0.8

4(1 2.6 ( 0.4 84 ( 1 0.5 ( 0.3 0 1.9 ( 1.1 0 5.6 ( 0.9

105 °C/24 h (oven-drying) incineration (525 °C/5 h) Kjedhal Soxhlet extraction (hexane) ADF-NDF ADF-NDF ADF-NDF UV spectrophotometry (Folin-Ciocalteu reagent)

moisture content (%) ash (%) proteins (F ) 6.25) lipids (%) cellulose (%) lignin (%) hemicellulose (%) phenolic compounds (%) a

SFPI ) sunflower protein isolate. b Averages and standard deviations of three experiments.

50 parts of water. In some cases, sodium sulfite (0-10 parts) was added. The mixture was then conditioned in an airtight container for 12 h at 25 °C. The equilibrated mixtures were then randomly used for rheological analysis or for the formation of injected objects. 2.2. Rheological Study. A Rheomex single-screw extruder (Haake Polylab System, Karlsruhe, Germany) equipped with a capillary die (L/D ) 10, D ) 3 mm) was used for rheological studies. The temperature of the die was between 100 and 150 °C. The temperature of each unit of the barrel was 10 °C lower than that of the previous unit or die. The temperature of the supply tank was fixed at 50 °C for all measurements. The screw speed was between 20 and 200 rpm. The compression rate of the rheometer screw was 2. 2.3. Modeling Experimental Curves of Viscosity. The apparent shear rate for Newtonian fluids is calculated according to

γ ) 4Q/πRc3

(1)

where Q is the volumetric flow rate in cubic centimeters per second and Rc is the radius of the capillary equipping the die in centimeters. The shear stress of the wall is expressed as a function of the difference between the pressure at the beginning of the capillary and atmospheric pressure (∆P expressed in pascals), the length of the capillary Lc and the radius of the capillary Rc, both of which are expressed in centimeters

τp ) Rc∆P/2Lc

(2)

If the fluid is pseudoplastic, the shear rate must be corrected by the following equation, known as the Weissenberg-Rabinowitch equation

γp ) γ(3m + 1)/4m

(3)

m ) d(log τp)/d(log γ)

(4)

with

In our case, this correction was directly calculated by the rheometer. For most thermoplastics, the shear stress is linked the shear rate by a power-law-type model

τp ) Kγm

(5)

The shear rate and shear stress can be used to obtain a curve showing the viscosity at the wall of the material tested by applying the following equation, known as the Ostwald-de Waele equation

η ) τp/γp ) Kγpm-1

(6)

The values of m are between 0 and 1 for thermoplastic materials, which explains their shear thinning behavior. The values of the coefficients K (consistency) and m (pseudoplasticity index) are obtained by linear regression of base-10 logarithm values of the real viscosity as a function of the base-10 logarithm values of the shear rate. This measurement, carried out in a single capillary, does not take edge effects into account and does not allow for the calculation of absolute values of viscosity. All of the viscosity values reported herein are therefore apparent viscosity values, as calculated from eq 6. 2.4. Thermal Study. A thermal study of the denaturation of mixtures elucidates their rheological behavior following an increase in the temperature of the barrel and die of the rheometer. The study was performed on a Pyris 1 power modulation differential scanning calorimeter (Perkin-Elmer), equipped with an Intracooler. The measurement cells were purged with dry nitrogen. The temperature was calibrated by use of indium (Tf ) 156.6 °C) and distilled water (Tf ) 0 °C). The capsules used for this study were airtight steel capsules with an O-ring. They resisted high pressure (40 bar) and were therefore well adapted for studying weakly hydrated proteins at high temperatures. The samples weighed approximately 10 mg. Each mixture was tested in triplicate. The measurements were taken during the first scan, at between 25 and 200 °C. The heating rate was 20 °C/min. 2.5. Production of Injection-Molded Samples. The equilibrated mixtures were molded with a Billion H280/90TP injection press (Oynnax, France). After numerous preliminary tests, the temperature profile selected was 50/100/110/120 °C for all mixtures. The resulting materials were in the form of standardized rectangular bars (80 mm × 10 mm × 4 mm) (ISO 294)12 and standardized dumbbells (150 mm × 10 mm × 4 mm) (ISO 527-2).13 After undergoing conditioning in humidity-controlled chambers until equilibrium [15 days at 43, 60, or 85% relative humidity (RH); 25 °C], the bars were subjected to flexural strength tests (ISO 178).14 The standardized dumbbells were used to measure the mechanical properties of the materials during tension tests (ISO 527-2).13 2.6. Mechanical Properties. A TA-XT2 texture analyzer (RHEO Stable Micro Systems, London, U.K.) was used to assess the flexural properties of the test specimens. The test samples were 80 mm long and 5 mm wide. Their thicknesses were measured at five points with a digital micrometer (model IDC-112B, Mitutoya Corp., Tokyo, Japan), and the mean values were recorded. Flexural stress was measured to determine the flexural strength at break (σmax) and elastic modulus (Ef). The grip separation was 50 mm, and the test speed was 5 mm‚min-1.

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An MTS 1/M (MTS Systems France, Cre´teil, France) apparatus was used for tension tests. The crosshead speed was 5 mm‚min-1, and the initial grip separation was 110 mm. The measurement of the force exerted by the apparatus as a function of elongation was used to determine the tensile strength (σmax), the strain at break (max), and the Young’s modulus (Ey) of the injected dumbbells. 3. Results and Discussion 3.1. Rheological Study. The aim of the rheological study was to evaluate the flow properties of different SFPI/glycerol/water mixtures. First, SFPI/water mixtures were tested, but without success. Even though they were easily fed, these mixtures did not result in a continuous and homogeneous flow. For the lowest moisture contents (mixtures of 100 parts of SFPI with 30, 20, and 10 parts of water), the mixture rapidly accumulated in the screw cover such that it could not be transported or made to flow through the die. At higher moisture contents (40 and 80 parts of water), this phenomenon was avoided, but the flow was disturbed by the evaporation of the water, which caused the material to expand and led to large variations in pressure at the outlet of the die. When the temperatures of the cover modules and dies were decreased, the amplitude of this phenomenon was decreased but to the detriment of the flow, which became problematic when the temperature of the die was below 100 °C. SFPI/glycerol mixtures were then studied in a second series of experiments. The difficulties encountered with these mixtures were due to feeding problems. Mixtures containing less than 50 parts of glycerol formed a compact powder that accumulated in the loading funnel, and this impaired feeding. Mixtures containing more than 50 parts of glycerol became thick, making feeding impossible. These first attempts led us to test SFPI/ glycerol/water mixtures. These mixtures were powdery and nonsticky, and allowed the screw to be continuously fed by gravity from the loading funnel. Thus, the first curves linking the viscosity of the mixtures to the shear rate were obtained. 3.1.1. Effect of Moisture Content. First, the amount of glycerol in the mixtures was fixed at 50 parts for 100 parts of SFPI, and the temperature of the die was set at 120 °C. The moisture content (including the moisture initially present in the SFPI) was between 10 and 50 parts. Figure 1 shows how the viscosity varies according to the moisture content. The viscosity decreased as the moisture content increased. This decrease was substantial between 10 and 20 parts of water and then more moderate as the moisture content increased further. This illustrates the plasticizing effect of water. These experimental data were analyzed using the power-law model (eq 6) linking viscosity to the shear rate. The results obtained (Table 2) demonstrate the shear thinning and thermoplastic nature of sunflower proteins and, therefore, their ability to flow under certain conditions. The coefficient m decreased from 0.58 to 0.24 as the moisture content increased from 10 to 50 parts. These findings are comparable to those obtained by Willett et al.15 for starch. These authors showed that the coefficient m decreased significantly (from 0.49 to 0.32) as the moisture content of a starch that had been destructured by extrusion at 15% RH increased from 15 to 30%.

Figure 1. Effect of moisture content and sodium sulfite content on the viscosity of mixtures of SFPI/glycerol/water/sodium sulfite ) 100/X/Y/x parts by weight. Die temperature ) 120 °C. Average and standard deviation values of three experiments were plotted Table 2. Viscosity Values for Mixtures of SFPI,a Glycerol, and Water (100/50/X Parts by Weight): Power-Law Modelb,c Coefficients as a Function of the Moisture Contentd moisture content (parts) coefficient

10

20

30

40

50

(Pa‚sm)

consistency K 35 701 33 024 29 338 26 466 24 485 m 0.58 0.34 0.30 0.29 0.24 correlation coefficient 0.9972 0.9982 0.9960 0.9908 0.9984 a SFPI ) sunflower protein isolate. b η ) Km-1, where m is the pseudoplasticity coefficient. c Averages and standard deviations of three experiments. d Conditions: SFPI content ) 100 parts, glycerol content ) 50 parts, barrel temperatures (°C) ) 50/100/ 110/120.

Figure 2. Effect of glycerol content on the viscosity of mixtures of SFPI/glycerol/water ) 100/X/Y parts by weight. Die temperature ) 120 °C. Averages and standard deviations of three experiments are plotted.

3.1.2. Effect of Glycerol Content. For this study, the moisture content was kept constant (50 parts), and glycerol contents of between 50 and 10 parts were tested. The temperature of the die was fixed at 120 °C. The viscosity decreased as the glycerol content increased (Figure 2). Furthermore, the coefficients of the powerlaw model (consistency, K, and pseudoplasticity, m) also decreased (Table 3). The coefficient m decreased from 0.36 to 0.24 as the glycerol content increased from 10

Ind. Eng. Chem. Res., Vol. 42, No. 8, 2003 1677 Table 3. Viscosity Values for Mixtures of SFPI,a Glycerol, and Water (100/X/50 Parts by Weight): Power-Law Modelb,c Coefficients According to the Glycerol Contentd glycerol content (parts) coefficient

10

20

30

40

50

(Pa‚sm)

consistency K 54 364 51 852 43 637 37 097 24 485 m 0.36 0.30 0.28 0.27 0.24 correlation coefficient 0.9925 0.9914 0.9906 0.9937 0.9984 a SFPI ) sunflower protein isolate. b η ) Km-1, where m is the pseudoplasticity coefficient. c Averages and standard deviations of three experiments. d Conditions: SFPI content ) 100 parts, moisture content ) 50 parts, barrel temperatures (°C) ) 50/100/ 110/120.

Figure 3. Effect of sodium sulfite content on the viscosity of mixtures of SFPI/glycerol/water/sodium sulfite ) 100/30/30/x parts by weight. Die temperature ) 120 °C. Averages and standard deviations of three experiments are plotted.

to 50 parts. Thus, this plasticizer has an effect similar to that of water in the mixture, but it does not affect the coefficient m as strongly, perhaps because it has a higher molecular weight. 3.1.3. Effect of Adding a Reducing Agent. The effect of adding sodium sulfite, a reducing agent that can be used to reduce the amount of plasticizer, was tested. Sodium sulfite can break inter- and intramolecular disulfide bridges and prevent them from reforming when the sample is heated as it passes through the screw die. Various amounts of sodium sulfite were added (1-10 parts) to a mixture of SFPI, glycerol, and water (100, 30, and 30 parts, respectively) (Figure 3). With 2 parts of sodium sulfite, the viscosity of the mixture was similar to that obtained with a mixture of 100 parts of SFPI, 50 parts of glycerol, and 50 parts of water (Figure 1). The viscosity was thus significantly lower. However, this phenomenon was only observed up to 4 parts of sodium sulfite (Figure 3). Beyond this optimum, the viscosity increased. Actually, the breaking of the intramolecular disulfide bonds at higher sulfite concentration might have led to greater unfolding and, hence, a more extended structure. Sodium sulfite might serve as a charge or as a complexing agent favoring the formation of a protein network and, therefore, increasing the viscosity of the mixture. 3.1.4. Effect of Temperature. The effect of temperature was studied on mixtures with a moisture content of between 10 and 50 parts and a constant glycerol content (50 parts). For moisture contents between 10 and 30 parts, the viscosity of the mixtures decreased

Figure 4. Effect of the temperature on the viscosity of mixtures SFPI/glycerol/water ) 100/50/Y parts by weight, where Y ) (a) 20, (b) 40, and (c) 50. Averages and standard deviations of three experiments are plotted.

as the temperature increased (Figure 4a). With 40 parts of water, the viscosity curves for the different temperatures were very similar, and the consistencies (K) obtained by modeling the experimental data increased with temperature (Figure 4b). This change in the behavior of the mixture was confirmed for a moisture content of 50 parts (Figure 4c). In this case, the viscosity increased with temperature. To further our understanding of this phenomenon, the temperature at which the sunflower proteins were denatured was recorded for mixtures with various moisture and glycerol contents (Figure 5). In the absence of glycerol, the denaturation

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Figure 5. Evolution of the denaturation temperature of mixtures of SFPI/glycerol/water ) 100/X/Y parts by weight, measured by DSC. Averages and standard deviations of three experiments are plotted.

Figure 6. Effect of temperature on the viscosity of mixtures of SFPI/glycerol/water/sodium sulfite ) 100/30/30/1 parts by weight. Averages and standard deviations of three experiments are plotted.

temperature decreased substantially (from 186 to 115 °C) as the moisture content increased (from 10 to 100 parts). This phenomenon was also observed, but to a lesser extent, in the presence of glycerol. For a constant content of 50 parts of glycerol, the denaturation temperature decreased from 137 to 115 °C as the moisture content increased from 10 to 100 parts. Conversely, at low moisture contents (i.e., 10 parts), increasing the amount of glycerol (0, 10, and 50 parts) significantly affected the denaturation temperature of the proteins (172, 164, and 137 °C, respectively). This phenomenon was less marked at 30 parts of water and almost undetectable at 50 parts. At the denaturation temperature, the three-dimensional structure of the protein is greatly modified: the protein chains unfold, thus exposing previously unexposed groups, and the chains acquire sufficient mobility and freedom to reorganize and form new bonds, such as disulfide bridges. This is known as coagulation,16 the extent of which is proportional to the temperature.17 Consequently, increasing the temperature of the die leads to more coagulated proteins and, thus, increases the viscosity. Conversely, below the denaturation temperature, an increase in temperature simply increases the molecular mobility and, therefore, decreases the viscosity of the mixtures studied. This thermal study improves our understanding of the rheological behavior of the mixtures. It was not possible, however, to compare the denaturation temperatures obtained by DSC directly with the temperatures recorded by the rheometer cells. The phenomenon of denaturation during the extrusion of mixtures through the single screw can also be affected by the presence of mechanical constraints that are absent in DSC. In zones subject to the strongest constraints, the local temperature can increase, and temperature probes do not reflect this phenomenon satisfactorily. Furthermore, errors due to the temperature being measured on the sheath (measurement taken on the wall and not in the center of the mixture) and the accuracy of these measurements themselves ((3 °C) further complicate the analysis. The effect of temperature was studied on mixtures containing sodium sulfite. For mixtures of SFPI, glycerol, and water (100, 30, and 30 parts by weight, respectively) containing 2 parts or more of sodium

sulfite, the viscosity decrease was such that, at 140 °C and above, it was virtually impossible to fill the screw homogeneously. The pressure measured at the outlet was less than 1 bar, and it was not possible to construct viscosity curves. At a lower sodium sulfite concentration (SFPI/glycerol/water/sodium sulfite ) 100/30/30/1 parts, Figure 6), an increase in temperature led to a decrease in viscosity, which is opposite to the findings in the absence of the reducing agent. This is consistent with the hypothesis that the presence of a reducing agent prevents the formation of disulfide bridges during extrusion. The flow of the mixture was therefore improved by increasing the temperature. Conversely, in the absence of the reducing agent, increasing the temperature favors the formation of covalent bonds, and this outweighs the natural increase in the fluidity of the mixture with temperature. 3.1.5. Effect of the Total Amount of Plasticizer Added. To optimize and thereby reduce the amount of plasticizer added (water and glycerol), tests were carried out using the optimal amount of sodium sulfite, i.e., 4 parts (Figure 7). The temperature of the die was fixed at 120 °C. The viscosity increased slightly as the amount of plasticizer decreased. The measurements were recorded without difficulty up to 15 parts of glycerol and 15 parts of water and validated the extrusion procedure for mixtures containing a reducing agent and small quantities of plasticizer. However, with less than 15 parts of glycerol and 15 parts of water, it was not possible to construct a satisfactory viscosity curve: the powder blocked the loading funnel, thus preventing correct feeding of the screw. 3.2. Injected Materials Made from Sunflower Proteins. Using the results of the rheological study, injection-molded objects were made from sunflower protein isolate. The composition of the injected mixture (SFPI/glycerol/water/sodium sulfite ) 100/18/18/4 parts by weight) was very similar to that shown to be optimal. The temperatures used were 50, 100, 110, and 120 °C for the screw die units, as determined in the preliminary assays. 3.2.1. Mechanical Properties of the Injected Samples. The samples obtained were shiny, smooth, and dark brown in color. When they came off the press, they were also relatively supple. As the diameter of the

Ind. Eng. Chem. Res., Vol. 42, No. 8, 2003 1679 Table 4. Compositions and Mechanical Characteristics of Injection-Molded Samples Produced from Sunflower Protein Isolate after Conditioning at 60% RH and 25 °C: Effect of Glycerol Contenta-c glycerol content (parts) average density

4 1.27 (0.02)

8 1.26 (0.02)

12 1.26 (0.02)

16 1.27 (0.03)

22 1.23 (0.01)

σmax (MPa) Ef (GPa)

41 (4) 2.32 (0.25)

flexural propertiesd 38 (3) 31 (1) 2.06 (0.04) 1.44 (0.09)

25 (2) 1.08 (0.14)

22 (1) 0.74 (0.05)

σmax (MPa) Ey (GPa) max (%)

16.1 (1.8) 2.00 (0.26) 0.58 (0.10)

tensile propertiese 14.5 (1.8) 1.88 (0.16) 0.79 (0.13)

11.6 (2.2) 0.93 (0.12) 1.21 (0.20)

10.6 (3.0) 0.50 (0.08) 1.79 (0.37)

13.2 (2.3) 1.35 (0.10) 0.93 (0.21)

a Averages and standard deviations (in parentheses) of seven experiments. b SFPI ) sunflower protein isolate. c Conditions: SFPI content ) 100 parts, moisture content ) 18 parts, barrel temperatures (°C) ) 50/100/110/120, injection pressure (kg‚cm-2) ) 1600, mold temperature ) ambient, Teflon mold. d Flexural properties: σmax, flexural strength; Ef, elastic modulus e Tensile properties: σmax, tensile strength; Ey, Young’s modulus; max, tensile strain.

Table 5. Compositions and Mechanical Characteristics of Injection-Molded Samples Produced from Sunflower Protein Isolate: Effect of the Relative Humidity during Conditioninga-c relative humidity (%)

43

60

85

22 (1) 0.74 (0.05)

4 (1) 0.06 (0.01)

propertiesd

σmax (MPa) Ef (GPa) σmax (MPa) Ey (MPa) max (%)

flexural 26 (2) 0.79 (0.04)

tensile propertiese 11.4 (3.7) 10.6 (3.0) 532 (95) 503 (86) 1.8 (0.2) 1.8 (0.5)

1.5 (0.2) 16 (1) 36.4 (9.6)

a Averages and standard deviations (in parentheses) of seven experiments. b SFPI ) sunflower protein isolate. c Conditions: SFPI content (parts) ) 100, moisture content (parts) ) 18, glycerol content (parts) ) 22, barrel temperatures (°C) ) 50/100/110/120, injection pressure (kg‚cm-2) ) 1600, mold temperature ) ambient, Teflon mold. d Flexural properties: σmax, flexural strength; Ef, elastic modulus e Tensile properties: σmax, tensile strength; Ey, Young’s modulus; max, tensile strain.

Figure 7. Effect of the total amount of plasticizers on the viscosity of mixtures SFPI/glycerol/water-sodium sulfite ) 100/X/Y/4 parts by weight, where X ) Y. Averages and standard deviations of three experiments are plotted.

loading funnel was larger than that of the rheometer, other mixtures, mostly containing less glycerol, could also be tested and injected. The compositions of the different mixtures and the mechanical characteristics of the injected pieces, conditioned at 60% RH and 25 °C, are reported in Table 4. The tensile strength at rupture decreased as the glycerol content increased (from 41 to 22 MPa for an increase from 4 to 22 parts of glycerol in 100 parts of SFPI). The findings for the elastic modulus were similar. These results simply demonstrate the plasticizing effect of glycerol, which increases as its concentration increases. The tension tests gave similar results. The maximal tensile strength (16.1 MPa) was observed with 4 parts of glycerol and decreased progressively to 10.6 MPa as the amount of glycerol increased to 22 parts. The elastic modulus of the injected pieces decreased from 2.0 to 0.5 GPa. The strain at break of the pieces was low, below 2 mm for the highest glycerol content (22 parts), which is 1.8% of the initial length of the sample. These results are similar to those obtained recently by Wang and Chen11 for injected materials made from soy protein. For an initial content of 100 parts of protein isolate, 13.5 parts of water, and 8 parts of glycerol, they reported a tensile strength of 12.9 MPa, a strain at break of 3.3%, and a Young’s modulus of 1.5 GPa. 3.2.2. Effect of Relative Humidity during Conditioning on the Mechanical Properties of the Injected Samples. All of the samples tested were made

from a mixture of SFPI, glycerol, water, and sodium sulfite (100, 22, 18, and 4 parts, respectively) and conditioned at 43, 60, or 85% relative humidity at 25 °C. The mechanical properties of the materials formed were strongly dependent on the relative humidity during conditioning (Table 5). The samples conditioned at 43% RH had mechanical properties similar to those of samples conditioned at 60% RH, although they were slightly more rigid. However, the properties were more different when the relative humidity during conditioning was 85%: the materials were much more supple. Their tensile strength was 10-fold lower, and their strain at break was 15-fold higher. This suggests that the materials are highly sensitive when the relative humidity is high. 4. Summary and Conclusions These studies describe the thermoplastic behavior of mixtures of sunflower protein isolate, glycerol, and water and demonstrate their suitability for the manufacture of injection-molded objects. Increasing the glycerol or water content increases the fluidity. A DSC study of the mixtures before injection provided evidence of the role of protein denaturation in the viscosity of these mixtures as the temperature increases. For mixtures containing 100 parts of SFPI, 50 parts of glycerol, and less than 40 parts of water, increasing the temperature improves the flow properties. This phenomenon is reversed when the water content is increased, probably as a result of a lowering of the denaturation temperature, which enables covalent bonds to form, to the detriment of the flow properties.

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The inclusion of a reducing agent significantly reduced the viscosity of these mixtures. It was thereby possible to make injected objects containing only small amounts of plasticizer. According to the standard test EN 13432, these objects are entirely biodegradable in liquid medium (mineralization yield ) 72% for 41 days of testing), which allows us to predict a complete and fast degradation in soil and in compost. In addition, these objects had good mechanical properties (σmax ) 16.1 MPa, max ) 0.6%, Ey ) 2.0 GPa for samples conditioned at 60% RH and 25 °C and made from a mixture of SFPI, glycerol, water, and sodium sulfite that was 100, 4, 18, and 4 parts by weight, respectively). However, their water resistance was low: at 85% RH, they absorbed more moisture than did the native isolate. The materials formed in this way are less elastic and less water-resistant than films previously obtained by thermo-molding.3 The network obtained by injection seemed to be less reticulated, possibly because of the presence of sodium sulfite in the mixture, but also because of the injection temperature. The temperature might not be sufficiently high to allow all potential covalent bonds to re-form after reduction. Were this the case, coagulation would not be complete, such that the properties of the injected materials would be poorer than those of thermo-molded films. Acknowledgment The authors thank the Agence de l’Environnement et de la Maıˆtrise de l’Energie (ADEME) for its financial support, which made this study possible. Literature Cited (1) Ayhllon-Meixueiro, F.; Vaca-Garcia, C.; Silvestre, F. Biodegradable films from isolate of sunflower (Helianthus annuus) proteins. J. Agric. Food Chem. 2000, 48, 3032-3036. (2) Orliac, O.; Rouilly, A.; Silvestre, F.; Rigal, L. Effects of additives on the mechanical properties, hydrophobicity and water uptake of thermo-moulded films produced from sunflower protein isolate. Polymer 2002, 43, 5417-5425. (3) Orliac, O.; Silvestre, F. New thermo-molded biodegradable films based on sunflower protein isolate: Aging and physical

properties. Presented at the 7th World Conference on Biodegradable Polymers and Plastics, Tirrenia (Pisa), Italy, Jun 4-8, 2002. (4) Shirai, M.; Okamura, K. Process for preparing fibrous protein food products. U.S. Patent 4,216,240, 1980. (5) Wittwer, F.; Tomka, I. Method for molding capsules. European Patent EP 0,090,600, 1987. (6) Tomka, I. Thermoplastic starch. In Water Relationships in Food; Levine, H., Slade, L., Eds.; Plenum Press: New York, 1991; pp 627-637. (7) Tomka, I.; Thoma, M.; Stepto, R. F. T. Shaped articles made from preprocessed starch. European Patent EP 0,304,401, 1989. (8) Lim, S.; Jane, J. Storage stability of injection-molded starch zein plastics under dry and humid conditions. J. Environ. Polym. Degrad. 1994, 2, 111-120. (9) Huang, H. C.; Chang, T. C.; Jane, J. Mechanical and physical properties of protein-starch based plastics produced by extrusion and injection molding. J. Am. Oil Chem. Soc. 1999, 76, 1101-1108. (10) Jane, J.; Wang, S. H. Soy protein-based thermoplastic composition for preparing molded articles. U.S. Patent 5,523,293, 1996. (11) Wang, S. H.; Chen, C.-H. Protein-based chewable pet toy. U.S. Patent 6,379,725, 2002. (12) ISO Standard. PlasticssInjection-Moulding of Test Specimens of Thermoplastic Materials; Reference ISO 294; International Organization for Standardization (ISO): Geneva, Switzerland, 1996. (13) ISO Standard. PlasticssDetermination of Tensile Properties. Part 2: Test Conditions for Moulding and Extrusion Plastics; Reference ISO 527-2; International Organization for Standardization (ISO): Geneva, Switzerland, 1993. (14) ISO Standard. PlasticssDetermination of Flexural Properties; Reference ISO 178; International Organization for Standardization (ISO): Geneva, Switzerland, 2001. (15) Willett, J. L.; Jasberg, B. K.; Swanson, C. L. Rheology of thermoplastic starch: Effect of temperature, moisture content, and additives on melt viscosity. Polym. Eng. Sci. 1995, 35, 202-210. (16) Boye, J. I.; Ma, C.-Y.; Harwalkar, V. R. Thermal denaturation and coagulation of proteins. In Food Proteins and Their Applications; Damodaran, S., Paraf, A., Eds.; Marcel Dekker: New York, 1996; pp 25-56. (17) Rouilly, A.; Orliac, O.; Silvestre, F.; Rigal, L. Thermal denaturation of sunflower globulins in low moisture conditions. Thermochim. Acta 2003, 398, 195-201.

Received for review November 14, 2002 Revised manuscript received February 7, 2003 Accepted February 11, 2003 IE020913X