Solution Blowing of Soy Protein Fibers - American Chemical Society

ARTICLE pubs.acs.org/Biomac

Solution Blowing of Soy Protein Fibers S. Sinha-Ray,† Y. Zhang,† A. L. Yarin,*,†,‡ S. C. Davis,§ and B. Pourdeyhimi§ †

Department of Mechanical and Industrial Engineering, University of Illinois at Chicago, 842 West Taylor Street, Chicago, Illinois 60607-7022, United States ‡ Center for Smart Interfaces, Technische Universit€at Darmstadt, Petersen str. 32, 64287 Darmstadt, Germany § 3427 The Nonwovens Institute, Box 8301, North Carolina State University, Raleigh, North Carolina 27695-8301, United States

bS Supporting Information ABSTRACT: Solution blowing of soy protein (sp)/polymer blends was used to form monolithic nanofibers. The monolithic fibers were blown from blends of soy protein and nylon-6 in formic acid. The sp/nylon-6 ratio achieved in dry monolithic nanofibers formed using solution blowing of the blend was equal to 40/60. In addition, solution blowing of core
shell nanofibers was realized with soy protein being in the core and the supporting polymer in the shell. The shells were formed from nylon-6. The sp/nylon-6 ratio achieved in dry core
shell fibers was 32/68. The nanofibers developed in the present work contain significant amounts of soy protein and hold great potential in various applications of nonwovens.

’ INTRODUCTION Modern technology is experiencing a significant shift toward biodegradable, renewable, or just “green” agricultural biomolecular materials. The shift is driven by consumer awareness, stringent regulations regarding synthetic waste, and a necessity to reduce dependence on synthetic materials. As a result, significant efforts are directed toward production of biodegradable materials from cotton, corn, potato,1
5 and soy protein and development of biodegradable composites.6
8 In particular, there is a growing emphasis on making soy-based “green materials”. The reason behind the drive toward soy-based materials can be attributed to the fact that soy protein is one of the cheapest and most widely grown vegetable products with a wide range of uses. Booming SoyDiesel production9,10 facilitates increasing production of soy while using only soy oil and leaving behind abundant residual soy protein. In addition to the use of soy protein as a nutrient, it has great industrial value too. Soy protein contains sialic acid,11 which is able to inhibit or capture influenza virus as receptor. Soy protein can be used as plastics, adhesives, and fire retardants.12
14 As a result of all of these advantages, there is a growing need and market for nanofibers containing soy protein isolates, utilizing their high surface area to volume ratio, which facilitates their functionality. Several attempts were undertaken to produce such fibers through electrospinning15
19 or wet spinning.20 In these works, electrospinning mostly relied on blends with a fiber forming polymer and soy protein because pure soy protein fibers are not flexible and it is very hard to achieve submicrometer fibers. Moreover, in such solution-based methods as electrospinning and wet spinning, dissolution of soy protein can be facilitated by denaturation by means of thermal treatment,20,21 alkali, for example, NaOH, r 2011 American Chemical Society

sodium sulfite, and urea,21,22 as well as pH and ionic strength variation.22 Note also that to improve tensile strength of protein/ polymer fibers, cross-linking was applied.20 It is emphasized that soy nanofibers were unavailable at present, as to our knowledge. The nanofiber market is growing. The soy functionality would add value to the growing nanofiber market. The nanofiber market is expected to reach 2.2 billion U.S. dollars; by 2020.24 In the present work, we employ a novel method, solution blowing,23 to produce monolithic and core
shell nanofibers containing soy protein. We denote as monolithic the fibers made of sp/polymer blends and distinguish them from core
shell fibers where sp is located in the core, and a polymer is predominantly in the shell. It is emphasized that in this work it was impossible to form pure sp fibers. Indeed, pure soy protein is very brittle and does not lend itself to fine fiber formation. In ref 15, electrospinning with pure sp was attempted and resulted in spraying only.

’ EXPERIMENTAL SECTION Materials. For solution blowing of monolithic and core
shell nanofibers containing soy protein, polyamide-6 (nylon-6) obtained from BASF (Mw = 65.2 KDa) was used. Nylon-6, a common industrial fiber-forming polymer, was chosen because it is soluble in formic acid, which is also a solvent for soy protein isolate, enabling the formation of homogeneous solution blends. The solvent used was formic acid, grade >95%, obtained from Sigma-Aldrich. The soy protein isolate PRO-FAM 955 (sp 955) was generously donated by ADM Specialty Food Received: March 30, 2011 Revised: May 6, 2011 Published: May 09, 2011 2357

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Figure 1. Shear viscosity versus shear rate of two solutions: (a) soy protein (sp 955) solution in formic acid and (b) sp 955 and nylon-6 solution in formic acid. Triangles correspond to the data acquired when ramping up in shear rate, whereas squares correspond to the data acquired when ramping down.

Figure 2. Panels (a) and (b) show SEM images of the monolithic fibers formed by blowing a blend of nylon-6 and soy protein PRO-FAM 955 in formic acid. The zoomed-in view of a section of (a) is shown in panel (b). The fibers are rather curly. Panel (c) shows for comparison fibers formed by blowing a blend of soy protein PRO-FAM 974 and nylon-6 in a 17/83 ratio in formic acid. The macroscale web shown in panel (d) corresponds to the nanofibers of panel (c).

Ingredients. It was used to obtain all results reported in this work except those in Figures 2c,d and 3, where soy protein isolate PRO-FAM 974 was used for comparison. PRO-FAM 955 and PRO-FAM 974 have the same composition but differ in the way they were processed, even though it might be expected that their denaturation in solution can make them practically indistinguishable. Both PRO-FAM 955 and PRO-FAM 974 were recommended by the United Soybean Board (USB) and furnished by ADM. All materials were used as received. For fluorescent imaging, the following materials were used: phosphate-buffered saline (PBS), bovine serum albumin (BSA; Sigma A7906), a rabbit antisoy antibody (S 2519; Sigma-Aldrich, Steinheim, Germany), and a goat antirabbit IgG FITC antibody (cat. no. 10004302, Cayman, Ann Arbor, MI). Solution Preparation. For solution blowing23 of monolithic nanofibers, blends of nylon-6 and sp 955 in formic acid were used. Solutions based on formic acid were prepared as follows. First, 1 g of soy

protein sp 955 was mixed with 8.5 g of formic acid and left on a hot plate at 60
65 °C for 24 h. Next, 1.5 g of nylon-6 was added to the solution. For comparison (cf. Figures 2 c,d and 3 below), the additional blends of soy protein sp 974 and nylon-6 in formic acid were prepared with the sp/nylon-6 ratio of 40/60 (w/w). It is emphasized that soy protein was completely dissolved in the acid. It should be mentioned that the pH of formic acid is 2, which is significantly lower than pH 4.5 corresponding to the isoelectric point of soy protein. The color of the soy protein solution changed to transparent brownish instead of opaque, which corresponds to a complete denaturation of soy protein, as explained in ref 25. Note also that for acidic solvents, solubility of 20% of a soy protein at pH 2 was reported in ref 21. That work also reported practically no solubility at pH ∼5. For the solution blowing of core
shell fibers,23 the shell was formed from a 20 wt % nylon-6 solution in formic acid. The core was prepared as follows: 1.3 g of sp 955 was completely dissolved in 8.7 g of formic acid. After that, 1 g of nylon-6 was also dissolved in that solution. The addition of nylon-6 to the core was also done to increase viscoelasticity, which facilitates solution blowing. It is emphasized that in the present work our aim was to achieve the highest possible content of soy protein in solution blown fibers. It was found that for monolithic fibers the maximum possible soy protein content in formic acid was 1/8.5, that is, 0.1176 g/g. Any further increase in the soy protein content disrupted the solution blowing process. In solution blowing of core
shell fibers, the jet was additionally stabilized by the polymer shell, which allowed a higher soy protein content in formic acid of 1.3/8.7, that is, 0.149 g/g. Rheological Characterization. Solutions used for blowing experiments were characterized rheologically in a simple shear experiment. The shear measurements were carried out using a Brookfield DV-IIþ PRO cone-and-plate viscometer. Figure 1 depicts the data corresponding to two solutions: (i) a solution of sp 955 in formic acid (1 g of sp 955 in 8.5 g of formic acid) and (ii) a solution of sp 955 and nylon-6 in formic acid (described in the previous subsection). In both cases, the flow curves show an expressed shear-thinning behavior, albeit shear viscosity of the sp solution is two to three orders of magnitude lower than that of the sp/nylon-6 solution. Setup and Experimental Procedure. The setup used for solution blowing in this work is described in detail elsewhere.23 In brief, to form monolithic fibers, sp/nylon-6 blend was supplied from a syringe pump into a reservoir attached to a core nozzle and issued from the latter. The core nozzle was surrounded by a concentric annular nozzle issuing a coaxial high speed nitrogen jet at speed of 230
250 m/s from a high pressure cylinder. The nitrogen jet accelerated and stretched the 2358

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Figure 3. EDS spectra of (a) cast PRO-FAM 974 soy protein film, (b) 40/60 sp/nylon-6 nanofibers with beads, (c) single bead from 40/60 sp/nylon-6 blend, (d) SEM image of fiber only region (box S3) used for EDS collection in e, and (e) nanofiber only region. core sp/nylon-6 jet. In the setup used for coblowing of core
shell nanofibers, two different core and shell solutions were supplied separately by two different syringe pumps into a coaxial core
shell nozzle. The core
shell polymer jet was issued inside a concentric outer nozzle, which sustained the nitrogen jet surrounding the core
shell sp/nylon-6 jet. Depending on the solutions used, minor adjustments were done in the size of the nozzle issuing them. In particular, a 13 gauge needle was used for blowing blends of soy protein with nylon-6 to prevent clogging. For blowing core
shell fibers, the needle sizes were initially chosen as in ref 23, where a 25 gauge needle was used as a core and a 16 gauge needle was used as a shell. The shell flow rate tested first was 5 mL/h, and the core flow rate was 1.6 mL/h. However, because the core diameter was relatively small, the flow in it was frequently interrupted and required cleaning. Then, it was established that the best choice for blowing core
shell fibers is to issue the shell solution through a 13-gauge needle and the core solution through an 18-gauge needle. These sizes were sufficient to prevent clogging and sustain a continuous blowing process. It is emphasized that similarly to ref 23, a continuous blowing process was achieved when the core needle did not protrude from the concentric

outer needle. In distinction from ref 23, no electric field was used in the present work. This can be attributed to the fact that in ref 23 the core was a poly(methylmethacrylate) (PMMA) (Mw = 996 KDa) solution, which was highly viscoelastic and required an extra pulling force (the electric force resulting from the Maxwell electric stresses). The core soy protein blends (even with a small amount of nylon-6 added) had a low enough elasticity for which the aerodynamic drag imposed by the blowing gas was sufficient to sustain a continuous process. For blowing of monolithic fibers, the flow rates of the blends in all cases were varied in the range of 4 to 5 mL/h, and the stagnation pressure of the compressed gas was varied in the range 20
25 bar. For blowing of nylon-6-based core
shell fibers, the shell flow rate was set as 5 mL/h. The flow rate of the core was kept constant at 6 mL/h, and the stagnation pressure of the compressed nitrogen was varied in the range of 20
25 bar. Optical Characterization. All optical observations were done using optical microscope Olympus BX-51. SEM images were obtained using JEOL 6320F. Fluorescence images were recorded using a Nikon microscope (Eclipse E800). 2359

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’ RESULTS AND DISCUSSION Blends of Soy Protein and Polymer: Monolithic Fibers. The fibers obtained from the blend of soy protein and nylon-6 are shown in Figure 2. They have no visible grains of protein because it was completely dissolved in formic acid. Note that the fibers in the SEM images in Figure 2 look rather curly, and some knobs are visible in Figure 2c, where another type of soy protein (PROFAM 974) was used for comparison. A macroscale web image corresponding to Figure 2c is shown in Figure 2d. In dry monolithic nanofibers formed using solution blowing of soy protein (PRO-FAM 955) blends with nylon-6, the protein/polymer ratio of sp/nylon 40/60 was achieved, as determined by the initial solution composition after the solvent had evaporated. SEM EDS Analysis of Nylon-6/Soy Protein Monolithic Blends. PRO-FAM 974 product data show nominal amino acid and other mineral contents, 0.62 wt % sulfur from cystine and methionine and the elements Na (1.2 to 1.6 wt %), K (0.024 to 0.3 wt %), Ca (0.05 to 0.15 wt %), P (0.7 to 1.0 wt %), Fe (0.008 to 0.013 wt %), and Mg (0.025 to 0.10 wt %). EDS spectra from the SEM image collection were employed to detect the unique atomic markers from soy protein isolate to differentiate from nylon-6 in a monolithic blend of nanofibers, such as that shown in Figure 2c. Figure 3a shows the EDS spectra for a formic acid solution cast film of soy protein isolate PRO-FAM 974. The unique markers, P and S, are clearly seen. An EDS spectrum of 40/60 sp/ nylon-6 monolithic blend nanofibers containing some beads is shown in Figure 3b. It is emphasized that the blend of sp/nylon-6 blown to form the fibers used in Figure 3 was absolutely clear, which is an indication that it was homogeneous and no undissolved protein grains were present. When such a blend is drawn and rapidly precipitated during the solution blowing process, solid nanofibers are formed. When some droplets are disconnected and emitted by high-speed gas stream, beads are formed. The unique markers (P, S) are clearly seen in the nanofiber, as was seen in the cast film. The quantities of S from the soy protein amino acids are comparable to theoretical values (0.6 wt % in pure sp and 0.2% in the 40/60 sp/nylon-6 blend), as shown in Table S1 (cf. Supporting Information). Note that Al was omitted in the analysis because it was due to the aluminum foil nanofiber collection substrate. An EDS spectrum was collected with SEM zoomed into a single bead, as shown in Figure 3c. The P and S peaks are clearly visible, indicating the presence of soy protein. Finally, the EDS spectra were collected with SEM zoomed to a nanofiber-only region, as shown in the box labeled S3 of Figure 3d (image) and e (spectra). P and S are again clearly visible at levels consistent with a monolithic blend (Table S2; cf. Supporting Information). These EDS spectral data suggest compositional uniformity of a monolithic sp/nylon-6 solution resulting in nanofibers and some beads with the same composition. The EDS analysis of the beads showed both S and P, which proves that soy protein precipitated in the beads. The EDS analysis of a visually fiber-only region (Figure 3e box labeled S3) clearly revealed P and S. In particular, such a region in box S3 in Figure 3d shows bundles of physically separated nanofibers with no matrix. This proves that the soy protein and nylon-6 coprecipitated to form monolithic fibers. In other words, the soy protein was mixed with nylon-6 in the nanofibers and was not lying in between the fibers. Core
Shell Nanofibers of Soy Protein and Polymer. The SEM images in Figure 4 show the cross-sectional view

Figure 4. Panels (a) and (b) show SEM images of core
shell fibers made with fully denaturated soy protein and nylon-6. The core is clearly visible in the cross-sectional view in panel (a) because of a significant electron contrast between soy protein and nylon-6. The core boundary in panel (a) is also traced by a white contour to make it more visible in printed reproduction and marked by the arrow. The side view (b) shows that the nylon-6 shell of this core
shell fiber is porous.

(Figure 4a) and side view (Figure 4b) of the nylon-6-based core
shell nanofibers. Denaturation of soy protein by formic acid results in complete solubilization of the former, whereas the evaporation of the latter after fibers were formed leaves a porous nylon-6 shell seen in Figure 4b. The mechanisms of pore formation in nanofibers were previously discussed in the context of electrospinning in refs 26 and 27. They can be attributed to such factors as direct permeation of the shell by vapor escaping from the core as well as to the breath figures, that is, solvent evaporation cooling, which triggers water droplet condensation onto nanofibers from the surrounding humid air. The image in Figure 4a shows a significant grayness gradient between the core and the shell, which is attributed to a significant electron contrast of soy protein in the core and nylon-6 in the shell. In dry core
shell nanofibers, the sp/nylon-6 ratio was 32/68, as determined by the initial solution compositions and the corresponding flow rates after the solvent had evaporated. Fluorescence Microscope Imaging. Fluorescence imaging was used to map soy protein inside fibers formed by solution blowing of blends of nylon-6 with sp in formic acid and core
shell fibers with sp in the core and nylon-6 in the shell. The fiber mats were washed twice with PBS and then blocked with PBS containing 3% BSA (500 μL per well) at room temperature for 30 min. Then, the fiber mat was washed twice by PBS. After that, a rabbit antisoy antibody at a dilution of 1:2000 in PBS was added overnight at 4 °C. Thereafter, the fiber mats were washed twice again with PBS; then, a goat antirabbit IgG FITC antibody (cat. no. 10004302, Cayman) was added at a dilution 1:100 in PBS for 1 h at room temperature. Finally, the fiber mats were washed twice with PBS. Fluorescence images were recorded using a Nikon microscope (Eclipse E800). Illuminating light was focused on the sample through a 20/0.75 NA objective or a 10/0.25 NA objective, and images were recorded using a CCD camera (CoolSnap fx, Roper Scientific, Tucson, AZ). Figure 5 demonstrates that soy protein is uniformly distributed in the fibers with the stained soy protein visible everywhere as green color. It is emphasized that soy protein can show some fluorescence under the illuminating light. However, staining is typically required for more reliable observation. Nylon-6 does not possess any fluorescence of its own and cannot be stained using the aforementioned procedure. 2360

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Figure 5. Optical image (panel a: black and white) versus fluorescence images (panel b: in color) of the fibers formed by solution blowing a blend of nylon-6 and soy protein in formic acid. Images (a) and (b) were taken exactly at the same place of a fiber mat. Both images were taken with a 100 microscope objective. The green color indicates the presence of soy protein.

Figure 6. Optical images (panels a and c: black and white) and fluorescence images (panels b and d: in color) of core
shell fibers with soy protein and a small amount of nylon-6 in the core and 20 wt % solution of nylon-6 in formic acid used to form the shell. Panels (a) and (b) corresponding to exactly the same place in the fiber mat were taken with a 40 microscope objective. Panels (c) and (d) corresponding to exactly the same place in the fiber mat were taken with a 100 microscope objective. Green fluorescence indicates the presence of soy protein.

The core
shell fibers are depicted in Figure 6. The images in Figure 6 reveal that the diameter of the green fluorescence fibers in panels (b) and (d) is smaller than the corresponding diameter of the same fibers in the black-and-white optical images in panels (a) and (c). In particular, in panel (a), the optical image of a fiber has a diameter of 1 μm, whereas the fluorescence image of the same fiber is only 0.4 μm in diameter. This proves that soy protein is, indeed, located in the core of the core
shell fibers as intended. It is emphasized that the fluorescent markers could easily reach the soy protein core via the nylon-6 shell through multiple pores clearly visible in Figure 4b and also reported previously for the other types of core
shell fibers.26,27 Statistical Image Analysis. Nonwovens produced by solution blowing are composed of the entangled individual fibers. They

practically never end until the process is stopped. In this sense, they are similar to electrospun nanofiber mats. Therefore, measurements of fiber lengths and aspect ratios are meaningless. Cross-sectional diameter can vary even along the individual fibers because of the effect of the turbulent pulsations in gas and elastic wave propagation.28,29 Therefore, measurements of fiber diameter distribution can be important for the structural characterization of the solution-blown nonwovens and their further applications. Figure 7 depicts the diameter distributions measured from SEM images of sp/nylon-6 fibers blown from blends, whereas Figure 8 contains the corresponding data for sp/nylon-6 core
shell fibers. It is emphasized that the blend-blown (monolithic) fibers all belong to the submicrometer range, whereas the core
shell fibers can involve infrequent outliers as large as 2 μm. Still, the core
shell fibers are submicrometer on average. 2361

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Figure 7. Diameter distribution in sp/nylon-6 blend solution blown nanofibers.

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antisoy antibody and secondary FITC antibody, soy protein has been successfully marked in the fibers. It was shown that in the blend-blown fibers soy protein was distributed everywhere in the fibers. These results were in agreement with EDS data from SEM, which indicated homogeneous sp/nylon-6 blends. In the core
shell fibers, soy protein was located only in the fiber cores, as designed. The monolithic (blend-blown) fibers all belong to the submicrometer range, whereas the core
shell fibers can involve infrequent outliers as large as 2 μm. Nevertheless, the core
shell fibers are submicrometer on average. The monolithic and core
shell nano- and microfibers containing significant amounts of soy protein developed in this work hold great potential as an effective method of utilizing of the one of the most abundant natural materials. In particular, it is foreseen as an effective method of utilization and valorization of residual material left by soy diesel production, which can be useful in various applications of nonwovens. Moreover, soy protein isolates (SPIs) used in the present work contain 90% protein, whereas soy protein concentrates (SPCs) contain 50
70% protein.30 The highest protein content soy product commercially available was used for this study. Therefore, the use of SPC was not investigated. In addition, note that the solution blown sp/nylon-6 nanofiber mats produced in the present work look stronger than many electrospun nanofiber mats reported so far31 and are not brittle. A detailed study of their tensile properties is underway and will be reported in future.

’ ASSOCIATED CONTENT

bS

Supporting Information. Elemental composition from EDS spectra in Figure 3b,e. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author Figure 8. Diameter distribution in sp/nylon-6 core
shell solution blown fibers.

*E-mail: [email protected]. Phone: þ1(312) 996-3472. Fax: þ1(312) 413-0447.

’ CONCLUSIONS Solution blowing of monolithic and core
shell soy protein
polymer fibers was demonstrated. The blend fibers were produced from soy protein and nylon-6. They had a protein/ polymer ratio of 40/60 in the dry sp/nylon-6 fibers. The core
shell fibers were produced from protein/nylon-6 in the core and nylon-6 in the shell. They had a protein/polymer ratio of 32/68 in the dry core
shell fibers. The individual fibers and their nonwovens were analyzed using optical and scanning electron microscopy (with EDS). Soy protein and nylon-6 provided a significant electron contrast to make the protein core clearly distinguishable from the polymer shell. In the present work, the monolithic nanofibers made from sp/nylon-6 blends were basically nonporous on the scale of 10
100 nm because the images on this scale (e.g., Figure 2c) do not resolve any visible porosity, which still might be present on the smaller scale. The core
shell sp/nylon-6 fibers had pores in the shell on the scale of 10 nm (Figure 4b). Porous shells probably allow low-molecularweight fluids to communicate with the core when core
shell fibers are submerged into them, which makes them attractive for many applications. In addition, using staining with primary rabbit

’ ACKNOWLEDGMENT This work was supported by a research contract with the United Soybean Board, Chesterfield, MO (research contract no. 0491). Dr. M. Cho is acknowledged for his help with fluorescence microscopy, and A. Kolbasov is acknowledged for technical assistance. ’ REFERENCES (1) Kanczler, J. M.; Ginty, P. J.; Bany, J. A.; Clarke, N. M. P.; Howdle, S. M.; Shakesheff, K. M.; Oreffo, R. O. C. Biomaterials 2009, 29, 1892–1900. (2) Yu, G.; Fan, Y. J. Biomater. Sci., Polym. Ed. 2008, 19, 87–98. (3) Greene, J. J. Polym. Environ. 2007, 15, 269–273. (4) Kim, C. W.; Frey, M. W.; Marquez, M.; Joo, Y. L. Polymer 2006, 47, 5097–5107. (5) Kim, C. W.; Frey, M. W.; Marquez, M.; Joo, Y. L. J. Polym. Sci., Part B: Polym. Phys. 2005, 43, 1673–1683. (6) Huang, X.; Netravali, A. A. N. J. Macromol. Sci., Part A: Pure Appl. Chem. 2008, 45, 899–906. (7) Huang, X.; Netravali, A. A. N. J. Compos. Sci. Technol. 2007, 67, 2005–2014. (8) Chabba, S.; Netravali, A. N. J. Mater. Sci. 2005, 40, 6275–6282. 2362

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