Biomacromolecules 2005, 6, 707-712
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Electrospun Fibers from Wheat Protein: Investigation of the Interplay between Molecular Structure and the Fluid Dynamics of the Electrospinning Process Dara L. Woerdeman,*,†,‡ Peng Ye,§ Suresh Shenoy,‡ Richard S. Parnas,§ Gary E. Wnek,‡,⊥ and Olga Trofimova| R&D Green Materials, LLC, 3701 Market Street, Suite 340, Philadelphia, Pennsylvania 19104, Department of Chemical Engineering, Virginia Commonwealth University, Richmond, Virginia 23298, Institute of Materials Science, University of Connecticut, Storrs, Connecticut 06269, and Applied Research Center, College of William & Mary, Newport News, Virginia 23606 Received September 3, 2004; Revised Manuscript Received October 27, 2004
In the present work, we demonstrate the ability to electrospin wheat gluten, a polydisperse plant protein polymer that is currently available at roughly $0.50/lb. A variety of electrospinning experiments were carried out with wheat gluten from two sources, at different solution concentrations, and with native and denatured wheat gluten to illustrate the interplay between protein structure and the fluid dynamics of the electrospinning process. The presence of both cylindrical and flat fibers was observed in the nonwoven mats, which were characterized using both polarized optical microscopy and field emission scanning electron microscopy. Retardance images obtained by polarized optical microscopy exhibited evidence of molecular orientation at the surface of the fibers. We believe that fiber formation by electrospinning is a result of both chain entanglements and the presence of reversible junctions in the protein, in particular, the breaking and reforming of disulfide bonds that occur via a thiol/disulfide interchange reaction. The presence of the highest molecular weight glutenin polymer chains in the wheat protein appeared to be responsible for the lower threshold concentration for fiber formation, relative to that of a lower molecular weight fraction of wheat protein devoid of the high molecular weight glutenin component. Denaturation of the wheat protein, however, clearly disrupted this delicate balance of properties in the experimental regimes we investigated, as electrospun fibers from the denatured state were not observed. Introduction In recent work it was demonstrated that wheat protein can be converted into a plastic material when it is chemically modified and compression-molded into some shape.1 A point of interest was whether the compression-molding step had led to an overall increase in the number of cross-links in the protein-based plastic. It has been postulated that the unfolding of the higher molecular weight glutenin proteins heated to 75 °C facilitates the sulfhydryl/disulfide interchange reactions between the exposed functional groups.2 The gliadin proteins (lower molecular weight protein fraction) were found to behave in a similar fashion at temperatures above 75 °C.2 Others have observed that heating of a gluten film-forming solution promotes protein chain interaction, giving rise to a film with enhanced mechanical properties.3 Electrospray ionization mass spectrometry (ESI-MS) has been used to study the reversibility of heat-induced conformational changes in proteins.4 The stability of a protein to * To whom correspondence should be addressed. E-mail:
[email protected]. † R&D Green Materials. ‡ Virginia Commonwealth University. § University of Connecticut. | College of William & Mary. ⊥ Present address: Department of Chemical Engineering, Case Western Reserve University, Cleveland, Ohio 44106.
denaturation is governed by factors such as the extent to which it is folded, the hydrophobicity of its core, the level of intramolecular hydrogen bonding in the system, and the density of disulfide bridges between individual polypeptide chains.4-7 In the ESI-MS study conducted by Mizra et al.,4 denaturation was found to lead to an elevation in the number of charges that attach to the protein. In general, an increase in the susceptibility of denatured protein chains to charging, as exhibited in the ESI-MS experiment, could lead one to assume that the denatured protein chains would be more likely to engage in acid-base interactions as they are unfolded and their polar groups become more accessible. While it appears that in certain materials applications it could be advantageous to denature the protein (by subjecting it to heat and/or pressure) as a means to improve the mechanical properties of the material, it is not clear if this would be the case in electrospinning, where fibers form quite readily as long as sufficient molecular entanglements exist among the polymer chains.8 In the present work, we explore the use of electrospinning as a way to engineer new material structures from commercial wheat protein, a bio-based polymer that has the important benefits of being both nontoxic and readily available.9 A descriptive model of wheat gluten proteins can be found in ref 1 and in references therein. It has also been observed that when wheat gluten is molded into plastic, a relatively stiff (E ≈ 1 GPa1 and 3.5
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GPa10) and strong (20-35 MPa1 and 50 MPa10) material results in comparison with other protein-based bioplastics.11 Electrospinning has gained popularity in the scientific community8,12-24 as a convenient method to produce nonwoven mats of micrometer- to nanometer-sized fibers. Feasibility experiments require only gram quantities of raw material. In the basic process, a reservoir of polymer fluid is charged and a fluid jet is accelerated through an electric field gradient toward a grounded target or collector. As the conical jet of polymer fluid propagates through the air, the solvent evaporates and a nonwoven mat of submicrometerdiameter fibers is produced on the collector. The electrospinning of various proteins, such as collagen12 and fibrinogen,13 has been demonstrated previously for potential use in biomedical applications, such as tissue scaffolds and hemostatic products. While these nonwoven mats exhibit stiffnesses (Young’s moduli) in the range of 25-55 MPa,12 it is unlikely that these materials will ever find use in any high-volume application due to cost alone (e.g., calfskin type I collagen is available at ca. $300/g). This current study on the electrospinning of commercial wheat protein comprises both a technological goal and a scientific goal, the technological goal being to test whether a nonwoven fibrous mat can be electrospun from a biopolymer as heterogeneous and polydisperse as commercial wheat gluten, while our scientific goal is to exploit our current understanding of electrospinning to elucidate the many complex interactions that occur in a wheat protein-based system. For example, researchers are currently working to establish guidelines for electrospinning in a variety of polymer systems, such as PVA.20 At a molecular weight of 9000-13000, the fibrous structure was not completely stabilized and a “bead-on-string” structure was obtained (except at 35 wt %), while a stable fibrous structure was successfully obtained as the molecular weight was increased to 13000-23000.20 Commercial wheat protein, on the other hand, contains a polymeric glutenin fraction (with molecular weights ranging from ca. 80000 to several million) and a monomeric gliadin fraction (MW < 50000),25,26 as well as a host of other constituents (starch and nonstarch polysaccharides, moistures, lipids, and minerals).1 In the present work, we leverage the results of recent electrospinning studies involving well-characterized, synthetic polymers, to enable us to better understand the electrospinning behavior and molecular-scale interactions of wheat gluten proteins. Experimental Section Materials. Two sources of commercial wheat gluten were used in this study: the first source was commercial wheat gluten [70.2% protein on an “as-is basis” as determined by the Dumas method (N × 5.7)] from Amylum Belgium N.V. (Aalst, Belgium), while the second source was commercial wheat gluten [75% protein (N × 5.7) as reported on the supplier “Technical Data” sheet] from MGP Ingredients, Inc. (Atchison, KS). Preparation of the Wheat Gluten Fraction. The commercial wheat gluten from Amylum Belgium N.V. was dispersed at room temperature at a 1:10 (w/v) ratio in 0.05
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M acetic acid. The dispersion was extracted for 1 h and centrifuged at 5000g for 15 min (T ) 20 °C). The pellet at the bottom of the centrifuge tube (high molecular weight component of gluten) was discarded. The supernatant was filtered and afterward spray-dried using a NIRO ATOMIZER spray dryer. The inlet and outlet temperatures were set at 130 °C and 108 °C, respectively. Spray drying of the supernatant resulted in the formation of a fluffy white powder comprising the low molecular weight protein fraction. Electrospinning. Wheat gluten was dissolved at concentrations between 5% and 10% (w/v) in 1,1,1,3,3,3-hexafluoro2-propanol (HFIP). Suspensions of wheat gluten were forced through a 5.0 mL syringe using a syringe pump (model 100, KD Scientific Inc., New Hope, PA), forming a bead of solution at the tip of the syringe. A high voltage was applied between the tip (an 18-gauge blunt needle) and a grounded collection target. In this particular experiment, the positive lead from a high-voltage supply (Spellman CZE1000R, Spellman High Voltage) was attached to the external surface of the metal syringe needle, and the rotating mandrel was placed 10-12 cm from the tip of the syringe. The syringe pump was set to deliver the protein suspension at rates up to 5 mL/h, and the applied voltage was fixed at 25 kV. Microscopic Analysis. Several specimens were analyzed by field emission scanning electron microscopy (FESEM), as well as by polarized optical microscopy. Samples were mounted onto SEM stubs, sputter-coated with AuPd, and examined using a Zeiss model DSM 982 FESEM microscope. Cross-sectional images were obtained by embedding the sample in epoxy. After the epoxy was cured, the block was cut using a glass blade to obtain a smooth surface. Crosssections were also prepared by “freeze fracture” in liquid nitrogen, or by simply cutting the material with a blade at room temperature. Traditional SEM was conducted using a Hitachi S-570 scanning electron microscope. In these experiments, samples were mounted onto SEM stubs and examined without further treatment. Results and Discussion Microscopic Characterization of the Electrospun Fiber Mats. The electrospun structure of several wheat proteinbased nonwoven mats was analyzed using traditional SEM, FESEM, and polarized optical microscopy. HFIP, a highly versatile and volatile solvent known for its ability to dissolve nylons and proteins,12 was employed in the present work. Specimen A depicts a nonwoven mat electrospun from asreceived wheat gluten that was provided by MGP Ingredients, Inc., an American supplier, while specimen B (Figure 1) was prepared under identical conditions using as-received commercial wheat gluten from Amylum Belgium N.V. (Table 1). Upon visual inspection, specimens A and B appear to be similar to one another, as both fabrics are composed of flat fibers with evidence of spherical domains and some beaded fibers in the mat, which was observed at two different magnifications (Figure 2). Specimen C (Figure 3, left), on the other hand, was prepared from an acetic acid-extractable fraction of wheat gluten, yielding a nonwoven mat with fewer
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Electrospun Fibers from Wheat Protein Table 1. Nonwoven Specimens Electrospun or Electrosprayed from Wheat Gluten specimen A specimen B specimen C specimen D
electrospun from as-received commercial wheat gluten from MGP Ingredients, Inc. electrospun from as-received commercial wheat gluten from Amylum Belgium N.V. electrospun from the 0.05 M acetic acid-extractable (low molecular weight) fraction of commercial wheat gluten from Amylum Belgium N.V. electrosprayed from denatured (temperature- and pressure-treated) commercial wheat gluten from Amylum Belgium N.V.
Figure 3. Scanning electron micrograph of an electrospun mat from the acetic acid-extractable fraction of commercial wheat gluten (left). Same electrospun fiber mat at a higher magnification (right).
Figure 1. Scanning electron micrograph of commercial wheat gluten electrospun onto a rotating, cylindrical mandrel at roughly 5000 rpm.
Figure 2. Scanning electron micrograph of electrospun commercial wheat gluten, exhibiting spherical domains among fibrils in the matrix (left). Nonwoven mat electrospun from commercial wheat gluten (right).
beads in the fibrous structure (Table 1). However, when viewed at high magnification (20000×), some debris can be seen at the surface of the fibers (Figure 3, right). Preliminary experimentation has shown that a concentration of 5% (w/v) of as-received wheat gluten in HFIP yields fibers during electrospinning (specimens A and B), while specimen C was formed by increasing the concentration of the wheat protein in solution to 10% (w/v). The solution concentration was increased to 10% (w/v) since a 5% (w/v) solution of the same acetic acid-extractable fraction of wheat protein yielded a spray rather than fibers. More specifically, the increase in wheat protein concentration is necessary since fractionation significantly lowers or eliminates the higher
molecular weight components.27 Consequently, specimen C is rich in the lower molecular weight gliadin proteins, and hence, a higher polymer concentration is required for fiber formation. Similar results have been reported for electrospinning of synthetic polymers.28,29 Additionally, specimen C is also relatively free of the nonproteinaceous constituents present in commercial-grade wheat gluten, such as starch and nonstarch polysaccharides, lipids, and minerals. The absence of both the highest and the lowest molecular weight components could explain the higher ratio of cylindrical fibers to flat fibers or “ribbons” in specimen C as compared to specimens A and B. Others have observed the preferential formation of flat fibers at higher concentrations and molecular weights.20,30 In the electrospinning of PVA,20 the authors identified a lower molecular weight regime that would yield cylindrical fibers and a higher molecular weight regime associated with the formation of flat fibers. In a material system as heterogeneous as wheat gluten, the wide array of geometries exhibited in Figures 1 and 2 could almost be anticipated. In addition to the broad range in features, ranging from circular to flat, ribbonlike fibers as depicted in a cross-sectional image of specimen B (Figure 4), we also see evidence of larger scale domains or “knots” distributed intermittently among the fibers, as described above (Figure 2). These knots could be indicative of the unstable fibrous “bead-on-string” structure that results when there is an insufficient number of molecular entanglements present in the polymeric network.31 An alternative explanation is that these domains are starch residues that have precipitated from the gluten solution. Fiber diameter distributions were analyzed from three low-magnification (1100×) images of each of the nonwoven specimens. All three specimens were found to have fiber distributions in the range of 100 nm to 5 µm, as illustrated in Figure 5. However, FESEM images of the fabrics acquired at very high magnifications (100000×) revealed the presence of even lower diameter fibers, as low as 25 nm.
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Figure 4. Cross-sectional image of a wheat gluten mat mounted in epoxy.
Figure 5. Normalized fiber size distribution as a function of fiber diameter group number for specimens A (hatched green bars), B (striped blue bars), and C (solid red bars). Group 1 corresponds to fiber diameters 3.0 µm. The statistics are only approximate as we counted only 40 fibers in each specimen, ignoring the small end of the distribution.
Physical Characteristics of the Electrospun Fibers. A polarized micrograph of the electrospun wheat gluten fibers in specimen B reveals another notable feature, namely, birefringence at the surface of the fibers (Figure 6). In particular, the retardance at the edge of the fibers is an indication of molecular orientation at the surface of the fibers. Muller et al.32 have previously reported a similar effect in high molecular weight atactic polystyrene (aPS) while investigating coil-stretch transitions under the influence of an elongational flow field. The authors demonstrated that at high strain rates (as applicable to electrospinning), network chain extension spreads out to streamlines along the periphery.32 As there is no evidence of birefringence inside the fibers, the electrospun fibers are believed to possess a type of “skin-core” structure, probably a result of the combined effect of solvent evaporation during the electrospinning process and the subsequent pulling of the electrospun fibers onto the rotating mandrel. The existence of a skin-core structure was corroborated by FESEM (Figure 7, left). (Figure 7, left) depicts a cross-sectional image of a flat fiber found in specimen B, and similar cross-sections can be
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Figure 6. Retardance image of an electrospun fiber mat acquired by polarized optical microscopy.
Figure 7. FESEM image of a flat fiber cross-section, depicting the fiber skin-core structure (left). FESEM image illustrating the pore structure on the surface of a fibril (right).
observed in specimens A and C. In all cases, the ribbons appear to be encapsulated by a skin layer. The surface of a micrometer-diameter fiber revealed the presence of 30 nm diameter pores along its length, as illustrated in Figure 7 (right). Similar microstructures have been reported on the surface of electrospun poly(lactic acid) (PLLA)33 and PS fibers.34 For PLLA,33 the authors attributed this to rapid phase separation during the electrospinning process, while fiber morphology in PS was determined to be a function of solvent volatility for PS fibers.34 Native vs Denatured Wheat Gluten. In the next phase of this study, the electrospinning technique was used as a method to compare the molecular characteristics of native wheat gluten with those of denatured wheat gluten. Size exclusion high-performance liquid chromatography (SEHPLC) measurements (Figure 8) of native and denatured wheat gluten appear to suggest an overall increase in crosslinking in a specimen that had been denatured via high heat (150 °C) and pressure (20 MPa).1 (For further details on how these data were obtained and analyzed, see ref 1.) In light of this, we set out to analyze the effects of these potential cross-links on electrospinning, particularly since it was unclear if the high viscosity introduced by the cross-links would impede the formation of electrospun fibers, even at high voltages. To test the fiber-spinning properties of
Electrospun Fibers from Wheat Protein
Figure 8. SE-HPLC chromatograph illustrating the differences in solubility of native wheat gluten versus heat- and pressure-treated wheat gluten. Increasing time on the x-axis inversely corresponds to decreasing molecular weight species eluting from the column. Gliadins and LMW-GS emerge from the column at 20-22 min, oligomeric proteins and HMW-GS emerge at 18-20 min, and extractable polymeric proteins (the highest molecular weight species) emerge from the column starting at 13 min. Compression molding appears to reduce the material’s overall solubility, which could be a physical manifestation of increased cross-linking in the denatured wheat gluten specimen. Adapted from ref 1.
denatured wheat protein, 5% (w/v) and 2.5% (w/v) solutions were prepared, among others, using compression-molded wheat protein ground to a fine powder using a mortar and pestle. The more dilute solution was prepared to compensate for the expected increase in solution viscosity, and electrospinning experiments were carried out using parameters identical to those used earlier. A variety of testing conditions were tried using a number of different concentrations of wheat protein in solution. Concentrations of 10% (w/v) and higher yielded a pastelike material and were therefore unusable in this experiment. The 5% and 2.5% (w/v) solutions, on the other hand, gave rise to an electrosprayed
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coating, as exhibited in the optical and scanning electron micrographs in Figure 9. Experiments involving the 5% (w/ v) solution were particularly noteworthy, since a portion of the protein suspension was clearly too viscous to overcome the resistance of surface tension. In short, it appeared as though the suspension had phase separated at the tip of the syringe; the low molecular weight (un-cross-linked) component led to the formation of the electrosprayed coating on the target (typically formed when the number of molecular entanglements is below some critical number), while the “cross-linked” component manifested itself in the form of centimeter-long fibers at the syringe tip. This result compelled us to further investigate why the native wheat gluten suspensions had yielded electrospun fibers so readily. Others35 have suggested that the polymeric component of gluten (glutenin) behaves like a cross-linked melt, where the viscoelastic properties are governed almost entirely by a combination of chain entanglements and the reversible junctions present in the system, namely, the breaking and re-forming of disulfide bonds via a thiol/ disulfide interchange reaction.2,25,35-37 According to Edwards et al.,35 the reversible formation of disulfide linkages in vital wheat gluten is more reminiscent of physical cross-links than that of traditional covalent linkages. In addition, the authors demonstrated that dough strength is directly correlated to chain extensibility and hence a function of the highest molecular weight fraction. Thus, clearly, the chemical interactions in wheat gluten are considerably more complex than those in synthetic polymer melts, the viscoelastic properties of which are usually governed by physical entanglements. In light of this, hydrated wheat gluten would perhaps be better regarded as a physical gel containing both reversible cross-links and entanglements. Taking into account the composition of the wheat protein, it is thus clear that fiber formation by electrospinning is a result of both chain entanglements38 and the presence of the reversible junctions. Similar results have also been obtained
Figure 9. Optical micrograph (100×) of the electrosprayed denatured wheat gluten coating (left). Scanning electron micrograph of the same specimen (1100×) (right).
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for synthetic polymer solutions, which are capable of physical gelation.39 The presence of the highest molecular weight fraction (glutenin) clearly lowers the threshold concentration (5 wt %) for fiber formation, as exhibited by specimens A and B. The acetic acid-extractable fraction (low molecular weight component) used to fabricate specimen C, on the other hand, had a slightly higher concentration threshold (10 wt %). In complete contrast to these results, fiber formation by electrospinning from the denatured protein solution was not observed. This could be related to the biochemical changes induced by the denaturation process. It is also well established that in the unfolded state sulfhydryl/disulfide interactions are facilitated, and addition of such chemical rearrangements will result in the “locking in” or stabilization of the denatured state.40 And we believe this phenomenon led to the two phases described earlier, namely, the viscous component, which proved incapable of forming electrospun fibers at 5% (w/v) concentration, and the predominately lower molecular weight component, which merely led to the formation of an electrosprayed coating at the applied voltage used in this experiment (25 kV). Summary We have succeeded in electrospinning nonwoven mats of wheat gluten ribbons and fibers with diameters ranging from several micrometers down to tens of nanometers. Polarized optical microscopy and FESEM revealed the presence of a skin layer on the surface of the fibers, with a porous microstructure. In developing these new material structures, we have also succeeded in shedding light on the behavior of wheat protein upon subjecting it to different processing environments. Finally, we have obtained additional evidence in support of our hypothesis that high-temperature processing of the wheat protein led to an increase in the number of crosslinks in the wheat protein-based molded product. Acknowledgment. D.L.W. is grateful for the technical assistance she received from Louis Krockaerts for setting up the spray dryer in the early stages of this study. D.L.W. and R.S.P. acknowledge the National Science Foundation (Grant No. DMI03306) for financial assistance. S.S. and G.E.W. would like to thank DARPA (Bio-Optic Synthetic Systems Program) and the NASA Office of Space Sciences for generous support. References and Notes (1) Woerdeman, D. L.; Veraverbeke, W. S.; Parnas, R. S.; Johnson, D.; Delcour, J. A.; Verpoest, I.; Plummer, C. J. C. Designing New Materials from Wheat Protein. Biomacromolecules 2004, 5, 12621269. (2) Schofield, J. D.; Bottomley, R. C.; Timms, M. F.; Booth, M. R. J. Cereal Sci. 1983, 1, 241-253. (3) Rayas, L. M.; Hernandez, R. J. U.S. Patent 6,045,868, Apr 4, 2000. (4) Mirza, U. A.; Cohen, S. L.; Chait, B. T. Anal. Chem. 1993, 65, 1-6. (5) Creighton, T. E. Proteins: Structures and Molecular Principles; W. H. Freeman: New York, 1984. (6) Ghelis, C.; Yon, J. Protein Folding; Academic Press: New York, 1982. (7) Lapanje, S. Physical and Chemical Aspects of Proteins Denaturation; Wiley-Interscience: New York, 1978.
Woerdeman et al. (8) McKee, M. G.; Wilkes, G. L.; Colby, R. H.; Long, T. E. Macromolecules 2004, 37, 1760-1767. (9) New and Improved Wheat Uses Audit. Prepared for the National Association of Wheat Growers by Sparks Companies, Inc., September 2002; available at http://www.wheatworld.org/html/news.cfm?ID)170. (10) The smaller values were obtained from tensile data collected at a low strain rate of 0.1 min-1 (erroneously reported as 10 s-1 in ref 1), while the larger values were obtained using a three-point bend test according to ASTM standard D790-02. We believe that neither set of values is more correct than the other. Rather, the discrepancy in the mechanical values is a reflection of normal polymeric timedependent behavior. (11) Mohanty, A. K.; Liu, W.; Tummala, P.; Drzal, L. T.; Misra, M.; Narayan, R. Soy Protein Based Plastics, Blends, and Composites. In Natural Fibers, Biopolymers and their Biocomposites; Mohanty, A. K., Misra, M., Drzal, L. T., Eds.; CRC Press Books: Boca Raton, FL, in press. (12) Matthews, J. A.; Wnek, G. E.; Simpson, D. G.; Bowlin, G. L. Biomacromolecules 2002, 3, 232-238. (13) Wnek, G. E.; Carr, M. E.; Simpson, D. G.; Bowlin, G. L. Nano Lett. 2002, 3 (2), 213-216. (14) Taylor, G. Proc. R. Soc. London, A 1969, 313, 453-475. (15) Formhals, A. U.S. Patent 1,975,504, 1934. (16) Arcidiacono, S.; Mello, C. M.; Butler, M.; Welsh, E.; Soares, J. W.; Allen, A.; Ziegler, D.; Laue, T.; Chase, S. Macromolecules 2002, 35, 1262-1266. (17) Ma, P. X. Mater. Today 2004, May, 30-40. (18) Um, I. C. Fang, D.; Hsiao, B. S.; Okamoto, A.; Chu, B. ElectroSpinning and Electro-Blowing of Hyaluronic Acid. Biomacromolecules, in press. (19) Casper, C. L.; Stephens, J. S.; Tassi, N. G.; Chase, D. B.; Rabolt, J. F. Macromolecules 2004, 27, 573-578. (20) Koski, A.; Yim, K.; Shivkumar, S. Mater. Lett. 2004, 58, 493-497. (21) Theron, S. A.; Zussman, E.; Yarin, A. L. Polymer 2004, 45, 20172030. (22) Yao, L.; Haas, T. W.; Guiseppi-Elie, A.; Bowlin, G. L.; Simpson, D. G.; Wnek, G. E. Chem. Mater. 2003, 15, 1860-1864. (23) Fong, H.; Chun, I.; Reneker D. H. Polymer 1999, 40, 4585. (24) Pedicini, A.; Farris, R. J. Polymer 2003, 44, 6857-6862. (25) Hamer, R. J.; Vliet, T. V. Understanding the Structure and Properties of Gluten: An Overview. In Wheat Gluten; Shewry, P. R., Tatham A. S., Eds.; The Royal Society of Chemistry: Cambridge, U.K., 2000; pp 125-131. (26) Singh, N. K.; Donovan, G. R.; Batey, I. L.; MacRitchie, F. Cereal Chem. 1990, 67, 150-161. (27) Be´rot, S.; Gautier, S.; Nicholas, M.; Godon, B.; Popine´au, Y. Pilot Scale Preparation of Wheat Gluten Protein Fractions I: Influence of Process Parameters on Their Protein Compositions. Int. J. Food Sci. Technol. 1994, 29, 489-502. (28) Huang, A. M.; Zhang, Y. Z.; Kotaki, M.; Ramakrishna, S. Compos. Sci. Technol. 2003, 63, 2223-2253. (29) Frenot, A.; Chronakis, I. S. Curr. Opin. Colloid Interface Sci. 2003, 8, 64-75. (30) Koombhongse, S.; Liu, W.; Reneker, D. H. Flat polymer ribbons and other shapes by electrospinning. J. Polym. Sci, Part B: Polym. Phys. 2001, 39 (21), 2598-2606. (31) Fong, H.; Chun, I.; Reneker, D. H. Polymer 1999, 40, 4585-92. (32) Muller, A. J.; Odell, J. A.; Keller, A. J. Non-Newtonian Fluid Mech. 1988, 30 99-118. (33) Bognitzki, M.; Czado, W.; Frese, T.; Schaper, A.; Hellwig, M.; Steinhart, M.; Greiner, A.; Wendorff, J. H. AdV. Mater. 2001, 13, 70-72. (34) Megelski, S.; Stephens, J. S.; Chase, D. B.; Rabolt, J. F. Macromolecules 2002, 35, 8456-8466. (35) Edwards, N. M.; Peressini, D.; Dexter, J. E.; Mulvaney, S. J. Rheol. Acta 2001, 40, 142-153. (36) Weegels, P. L.; Hamer, R. J.; Schofield, J. D. J. Cereal Sci. 1996, 23, 1-18. (37) Ewart, J. A. D. J. Sci. Food Agric. 1979, 30, 482-492. (38) Shenoy, S. L.; Bates, D.; Wnek, G. E. Polymer, submitted for publication. (39) Shenoy, S. L.; Bates, D.; Wnek, G. E. Polymer, submitted for publication. (40) Domenek, S.; Morel, M. H.; Moricel, J.; Guilbert, S. J. Agric. Food Chem. 2002, 50, 5947.
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