Plant proteins such as zein, wheat gluten, and soyproteins that are obtained as coproducts during processing of cereal grains have been used for industrial applications including fiber production. Although wheat gluten and soyproteins are available in larger quantities and have lower cost than zein, zein has been more extensively studied for fiber production since zein dissolves in aqueous ethanol solutions and has excellent spinnability. Although zein has excellent solubility in aqueous ethanol, the influence of various solvents on the electrospinnability and properties of fibers obtained was studied by Selling et al. [07Sel]. Table 59.1 shows the solvent, conditions used, and the properties of the fibers obtained. Lower alcohol/water solutions produced ribbonlike fibers, whereas acetic acid produced fibers with round morphology and narrower diameter distribution.

The plant protein zein was electrospun into fibers with the addition of 10 % chitosan to improve antimicrobial activity [09Tor]. Zein and chitosan were dissolved separately, mixed in 2:1 proportion, and electrospun into fibers with diameters in the submicron range. Although preferable, addition of higher than

Table 59.1 Comparison of the conditions used and properties of electrospun zein fibers [07Sel] Solvent Zein (%) Potential (kV) Distance (cm) Syringe pump flow rate (mL/h) Fiber morphology Fiber diameter (pm) 60% EtOH/ water 20 20 10 Gravity Ribbon with beads 230 60% EtOH/ water 27 20 10 Gravity Ribbon 160 70 % EtOH/ water 27 20 10 Gravity Ribbon 16 80% EtOH/ water 27 20 10 Gravity Ribbon 210 90% EtOH/ water 27 20 10 Gravity Ribbon 190 80% EtOH/ water 30 10 10 Gravity Ribbon 210 80% EtOH/ water 27 20 10 8 Ribbon 250 90% EtOH/ water 27 20 10 8 Ribbon 290 60% IPA/water 27 20 10 8 Ribbon 230 80% IPA/water 27 20 10 8 Ribbon 330 80% IPA/water 30 20 10 8 Ribbon 320 80% MeOH/ water 30 20 10 8 Ribbon 350 60% acetone/ water 27 20 10 8 Electrospray 60-90 % AcOH/ water 27 20 10 8 Electrospray AcOH 20­ 23 20 10 8 Electrospray — AcOH 27 20 10 8 Round 100 AcOH 30 20 10 8 Round 280 AcOH 30 20 10 4 Ribbon and round 220 (continued)

Table 59.1 (continued) Solvent Zein (%) Potential (kV) Distance (cm) Syringe pump flow rate (mL/h) Fiber morphology Fiber diameter (pm) AcOH 30 20 10 12 Round 400 AcOH 30 10 10 8 Round 220 AcOH 30 30 10 8 Round 280 AcOH 30 40 10 8 Round 240 AcOH 30 20 5 12 Round 410 AcOH 30 20 20 12 Round 270 EtOH is ethanol, MeOH is methanol, and AcOH is acetic acid

10 % chitosan resulted in the formation of excessive beads and it was not possible to obtain fibers. It was reported that addition of low amounts of chitosan provided substantial antimicrobial activity to the fibers. However, the stability of the fibers in aqueous media was not reported and no cross-linking was done. Ultrafine protein fibers with diameters between 150 and 600 nm were obtained from corn zein under the optimum conditions of 20 % protein, 70 % ethanol concentration, and voltage of 15 kV. Potential of the zein nanofiber mats to immobilize a plant polyphenol epigallocatechin gallate (EGCG) was investigated. Freshly spun fibers provided a relatively low immobilization power of 82 % compared to 98 % for fibers aged at 0 % relative humidity for 1 day. The electrospun fibers were considered to be suitable for encapsulation of biomolecules for food applications [09Li].

Although zein is easily electrospinnable, matrices developed from zein have poor aqueous stability and disintegrate upon immersion in aqueous media. To overcome this limitation, cross-linking of zein has been considered. In one such effort, zein was dissolved in acetic acid and glyoxal in various extents was added as the cross-linking agent. Optimizing conditions during electrospinning resulted in the production of fibers with diameters ranging from 0.3 to 67 pm [12Sel]. However, the stability of the matrices in various media was not studied. Cross-linking agents such as glyoxal and glutaraldehyde used to cross-link zein provide good water stability but are cytotoxic. To develop water stable and cytocompatible zein nanofibers, citric acid was used as the cross-linking agent. Both dry and wet cross-linking methods were developed to obtain electrospun matrices with desired properties. Uncross-linked matrices lost their morphology and became film-like when immersed in PBS (Fig. 59.2, left), whereas the cross-linked matrices were stable and retained their fibrous morphology even after 24 days (Fig. 59.2, middle). The cross-linked matrices were biocompatible and showed better potential for cell growth and proliferation (Fig. 59.2, right) than similar electrospun poly(lactic acid) matrices [10Jia, 12Jia].

To study the potential of using zein fibers for controlled release applications, three common (a, p, y) cyclodextrins were added into zein solution (in dimethyl formamide) to act as inclusion complexes which could attract and load biomolecules and electrospun into fibers with diameters between 100 and 400 nm

Fig. 59.2 SEM images show the un-crosslinked zein fibers lose their fibrous morphology and become film-like (left) when immersed in PBS at 37 °C for 2 days, whereas citric acid cross-linked zein fibers retain their fibrous morphology (middle) even after being in 37 °C PBS for 24 days. Confocal image depicting the growth of f-actin (red) on the fibrous zein scaffolds indicating biocompatibility (right)

[12Kay]. Table 59.2 provides a comparison of the properties of the fibers obtained with various concentrations of zein in solution and different amounts of cyclodex­trin [12Kay]. Similarly curcumin, a natural antimicrobial agent, was added into zein and electrospun into fibers with average diameters of 310 nm. Addition of curcumin increased the fluorescence and in vitro degradation studies showed sustained release of curcumin and retention of free radical scavenging ability [12Bra].

In addition to zein, other plant proteins such as soyproteins and wheat gluten and gliadin have been made into regenerated films, fibers, and other materials. Since these non-prolamin proteins do not dissolve in electrospinnable solvents, it is difficult to produce electrospun fibers. However, some reports are available on electrospinning wheat gluten and soyproteins. For instance, ability of producing electrospun fibers from native and denatured wheat gluten was examined by Woerdeman [05Woe]. In another report, wheat gluten was mixed with poly(vinyl alcohol) (PVA), dithiothreitol (DTT), and thiolated poly vinyl alcohol (PVA) in water/propanol and electrospun into fibers [10Don].

In a unique approach, soyproteins were extracted using urea and reducing agents and the reduced soyproteins obtained were dissolved using aqueous buffers containing surfactants. The soyprotein solution could be electrospun into 3D fibrous scaffolds that supported the attachment, growth, proliferation, and differentiation of stem cells [13Cai]. Figure 59.3 shows an image of the 3D fibrous soyprotein scaffold developed using the novel approach.

Table 59.2 Electrospinning conditions and properties of electrospun zein fibers obtained [12Kay] Solution % Zein (W/V) Viscosity (PaS) Conductivity (pS/ cm) Fiber diameter (nm) Zein 40 40 0.0332 435 — Zein 50 50 0.0859 344 80 ± 35 Zein 60 60 0.206 264 170 ± 30 Zein 40/a-CD10 40 0.0421 359 — Zein 40/P-CD10 40 0.0428 357 — Zein 40/Y-CD10 40 0.0439 333 60 ± 10 Zein 40/a-CD10 40 0.0522 270 60 ± 20 Zein 40/P-CD10 40 0.0562 283 70 ± 20 Zein 40/Y-CD10 40 0.0732 267 60 ± 10 Zein 40/a-CD10 40 0.0849 96.8 — Zein 40/P-CD10 40 0.0727 78.8 — Zein 40/Y-CD10 40 0.101 115.6 — Zein 40/a-CD10 50 0.125 286 90 ± 20 Zein 40/P-CD10 50 0.171 278 100 ± 25 Zein 40/Y-CD10 50 0.212 268 110 ± 30 Zein 40/a-CD10 50 0.212 138 185 ± 45 Zein 40/P-CD10 50 0.208 167 150 ± 30 Zein 40//-CD10 50 0.239 161 155 ± 35 Zein 40/a-CD10 50 0.39 74.3 240 ± 85 Zein 40/P-CD10 50 0.381 97.8 360 ±140 Zein 40/Y-CD10 50 0.354 126.5 265± 110 Zein 40/a-CD10 60 0.329 211 225 ± 30 (continued)

Solution	% Zein (W/V)	Viscosity (PaS)	Conductivity (pS/ cm)	Fiber diameter (nm)
Zein 40/P-CD10	60	0.292	200	185 ± 40
Zein 40/Y-CD10	60	0.218	189.4	170 ± 40
Zein 40/a-CD10	60	0.69	89.8	375 ± 80
Zein 40/P-CD10	60	0.441	113	410± 130
Zein 40/Y-CD10	60	0.664	109.6	380 ± 240
Zein 40/a-CD10	60	1.56	41.6	—
Zein 40/P-CD10	60	1.02	85.6	—
Zein 40/Y-CD10	60	0.752	85.8	—

Table 59.2 (continued)

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