Corn zein undergoes conformational changes to higher ß-sheet content during its self-assembly in an increasingly hydrophilic solvent.

Viscoelasticity of corn zein is associated with the formation of ß-sheet secondary structures; however, studies of the fundamentals of this conformational change are limited due to zein insolubility and poor analytical resolution. Here, changes in soluble zein conformation were evaluated as the protein self-assembles in increasingly hydrophilic solvents to the concentration just before aggregation and precipitation. Circular dichroism spectra of zein showed that α-helix structures decrease in favor of random coil and ß-sheets with increases in water content in an ethanol-water system, similar to observations of zein when it becomes viscoelastic in dough systems. This was further supported by changes in Thioflavin T fluorescence emission spectra and intrinsic viscosity measurements. Two widely recognized molecular models for α-zein (hairpin and superhelical conformations) were tested at 75 and 45% ethanol concentration using molecular dynamics simulation for agreement with experimental results. Increase in solvent hydrophilicity increased ß-sheets and reduced distance between backbone anomeric carbons only for hairpin model, suggesting it to be the more valid of the two. These findings emphasize the importance of transformation to ß-sheets during zein self-assembly and provide further insight into the mechanisms by which the protein is functionalized into viscoelastic systems.

Effects of plasticizer type and concentration on rheological, physico-mechanical and structural properties of chitosan/zein film.

The purpose of this study was to evaluate the effect of plasticizer type (glycerol, PEG-400, and sorbitol) and concentration (0%, 15%, 30% and 45%, w/w dry polymer weight) on rheological and physico-mechanical and structural properties of chitosan/zein blend film. Based on the analysis of rheological properties of chitosan/zein film-forming solutions, all film-forming solutions exhibited non-Newtonian behavior. The flow index of film-forming solution increased and apparent viscosity decreased with the increase of plasticizer concentration. The storage modulus (G') and the loss modulus (G″) decreased when plasticizer was added. The permeability of films increased significantly with the increase of plasticizer concentration, but the C/Z-P film (plasticized chitosan/zein film with PEG-400) had better barrier performance compared with the other two. The C/Z-P film had better mechanical properties and light transmission. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) revealed chitosan and zein had good compatibility due to the addition of the plasticizer, and crystallinity decreased with the increase of plasticizer concentration.

Effect of zein protein and hydroxypropyl methylcellulose on the texture of model gluten-free bread.

The influence of zein protein and hydroxypropyl methylcellulose (HPMC) on the texture and volume of gluten-free bread was investigated. The addition of HPMC to starch affected the dough viscoelasticity and it improved the bread volume during baking since it acts as an emulsifier. The addition of zein protein to gluten-free bread increased the crumb firmness and reduced the crust hardness within the range of concentrations investigated. No zein protein network could be observed in the bread crumb. The zein protein, cold mixed at low concentration, did not enhance the dough elasticity. Due to the lack of a protein network noncovalent interactions may stabilize the bubble structure stabilization within the crumb, rather than covalent links of the protein chain. With an optimized amount of zein protein and HPMC hydrocolloid, the gluten-free bread showed similar texture and staling behavior to that of model wheat bread. The optimized recipe, compiled into a spreadsheet, is available in the supporting information. The microstructural observations suggest that zein could be replaced with another protein for this recipe resulting in a similar bread texture.

OS1 functions in the allocation of nutrients between the endosperm and embryo in maize seeds.

Uncovering the genetic basis of seed development will provide useful tools for improving both crop yield and nutritional value. However, the genetic regulatory networks of maize (Zea mays) seed development remain largely unknown. The maize opaque endosperm and small germ 1 (os1) mutant has opaque endosperm and a small embryo. Here, we cloned OS1 and show that it encodes a putative transcription factor containing an RWP-RK domain. Transcriptional analysis indicated that OS1 expression is elevated in early endosperm development, especially in the basal endosperm transfer layer (BETL), conducting zone (CZ), and central starch endosperm (CSE) cells. RNA sequencing (RNA-Seq) analysis of the os1 mutant revealed sharp downregulation of certain genes in specific cell types, including ZmMRP-1 and Meg1 in BETL cells and a majority of zein- and starch-related genes in CSE cells. Using a haploid induction system, we show that wild-type endosperm could rescue the smaller size of os1 embryo, which suggests that nutrients are allocated by the wild-type endosperm. Therefore, our data imply that the network regulated by OS1 accomplishes a key step in nutrient allocation between endosperm and embryo within maize seeds. Identification of this network will help uncover the mechanisms regulating the nutritional balance between endosperm and embryo.

Structural characterization and formation mechanism of zein-propylene glycol alginate binary complex induced by calcium ions.

Effect of calcium ions (Ca2+) on characteristics of zein-propylene glycol alginate (PGA) binary complex was studied in this work. Ca2+ induced the formation of zein aggregates with decreased fluorescence intensity and a significant α-helix loss of zein. Zein-PGA binary complex with Ca2+ showed the decreased dimension and the minimum size was observed at 50.0mM Ca2+. Ca2+ resulted in the formation of strong hydrogen bonds between zein and PGA, strengthened their hydrophobic interactions, and induced a new peak at the diffraction angle of 30° in the pattern of Zein-PGA binary complex. PGA fortified with Ca2+ exhibited an overall plane-like structure, also an interwoven flat profile appeared in Zein-PGA binary complex with Ca2+. Three potential mechanisms were proposed to explain the morphological changes of samples after Ca2+ addition: (i) particle-particle collision and aggregation of particles; (ii) chain-chain association and further cross-linking of associated chains; (iii) simultaneous cross-linking coupled with aggregation.

Enhancement of mechanical properties, microstructure, and antimicrobial activities of zein films cross-linked using succinic anhydride, eugenol, and citric Acid.

Zein constitutes about half of the endosperm proteins in corn. Recently, attempts have been made to utilize zein for food coatings and biodegradable materials, which require better physical properties, using chemical modification of zein. In this study, zein proteins were modified using citric acid, succinic anhydride, and eugenol as natural cross-linking agents in the wet state. The cross-linkers were added either separately or combined in increment concentrations (0.1, 0.2, 0.3, and 0.4%). The effects of those agents on the mechanical properties, microstructure, optical properties, infrared (IR) spectroscopy, and antibacterial activities of zein were investigated. The addition of cross-linking agents promoted changes in the arrangement of groups in zein film-forming particles. Regarding the film properties, incorporation of cross-linking agents into zein films prepared in ethanol resulted in two- to three-fold increases in tensile strength (TS) values. According to the Fourier-transform infrared (FTIR) spectra and Hunter parameters there were no remarkable changes in the structure and color of zein films. Transparency of zein films was decreased differentially according to the type and cross-linker concentration. The mechanical and optical properties of zein films were closely related to their microstructure. All cross-linked films showed remarkable antibacterial activities against Bacillus cereus ATCC 49064 and Salmonella enterica ATCC 25566. Food spoilage and pathogenic bacteria were affected in a film-dependent manner. Our experimental results show that even with partial cross-linking the mechanical properties and antipathogen activities of zein films were significantly improved, which would be useful for various industrial applications.

Recent advances in food-packing, pharmaceutical and biomedical applications of zein and zein-based materials.

Zein is a biodegradable and biocompatible material extracted from renewable resources; it comprises almost 80% of the whole protein content in corn. This review highlights and describes some zein and zein-based materials, focusing on biomedical applications. It was demonstrated in this review that the biodegradation and biocompatibility of zein are key parameters for its uses in the food-packing, biomedical and pharmaceutical fields. Furthermore, it was pointed out that the presence of hydrophilic-hydrophobic groups in zein chains is a very important aspect for obtaining material with different hydrophobicities by mixing with other moieties (polymeric or not), but also for obtaining derivatives with different properties. The physical and chemical characteristics and special structure (at the molecular, nano and micro scales) make zein molecules inherently superior to many other polymers from natural sources and synthetic ones. The film-forming property of zein and zein-based materials is important for several applications. The good electrospinnability of zein is important for producing zein and zein-based nanofibers for applications in tissue engineering and drug delivery. The use of zein's hydrolysate peptides for reducing blood pressure is another important issue related to the application of derivatives of zein in the biomedical field. It is pointed out that the biodegradability and biocompatibility of zein and other inherent properties associated with zein's structure allow a myriad of applications of such materials with great potential in the near future.

Cell adhesion on zein films under shear stress field.

Vascular implants after implantation need to improve the ability of cells to withstand flow-shear stress. As such, we want to test whether zein films could improve the flow-shear stress resistance of cells by control of their surface morphology. We chose Collagen, poly L-lactic acid (PLLA) and three types of zein as the coating films and evaluated the flow-shear stress resistance of NIH3T3, and EA.hy926 on these respective films. The results showed that the retention of two cell lines on Collagen film was better than PLLA and zein films. The cell retention of EA.hy926 on Zein 3 film with higher roughness was better than Zein 1 film with a flat surface in the first 2h. The cell retention of NIH3T3 on a rougher surface was always better than the smoother one under flow-shear stress condition for 6h. Observation of cell morphologies showed that the aspect ratio changed significantly for NIH3T3 cells upon flow-shear stress condition, as shown by reduced numbers of pseudopodia, increased cell rounding and shrinkage. Zein 3 film with higher roughness improved the flow-shear stress resistance of cells and might be used in vascular implant coatings.

Effect of γ-zein on the rheological behavior of concentrated zein solutions.

Zein, the prolamin of corn, is attractive to the food and pharmaceutical industries because of its ability to form edible films. It has also been investigated for its application in encapsulation, as a drug delivery base, and in tissue scaffolding. Zein is actually a mixture of proteins, which can be separated by SDS-PAGE into α-, ß-, γ-, and δ-zein. The two major fractions are α-zein, which accounts for 70-85% of the total zein, and γ-zein (10-20%). γ-Zein has a high cysteine content relative to α-zein and is believed to affect zein rheological properties. The aim of this study was to investigate the effect of γ-zein on the often observed phenomena of zein gelation. Gelation affects the structural stability of zein solutions, which affects process design for zein extraction operations and development of applications. The rheological parameters, storage modulus (G') and loss modulus (G″), were measured for zein solutions (27% w/w solids in 70% ethanol). ß-Mercaptoethanol (BME) was added to the solvent to investigate the effect of sulfhydryl groups on zein rheology. Modulus data showed that zein samples containing γ-zein had measurable gelation times under experimental conditions, contrary to samples with no γ-zein, where gelation was not detected. Addition of BME decreased the gelation time of samples containing γ-zein. This was attributed to protein unfolding. SEM images of zein microstructure revealed the formation of microspheres for samples with relatively high content of α-zein, whereas γ-zein promoted the formation of networks. Results of this work may be useful to improve understanding of the rheological behavior of zein.

Green microfluidics made of corn proteins.

Petroleum-based polymer such as Poly(dimethylsiloxane) has been widely used to make mesoscale and microscale fluidic devices. The main drawback of such devices in disposable applications is the potential environmental pollution since they are not biodegradable. Biodegradable microfluidic devices have been fabricated out of zein, a prolamin protein found in corn, that can be utilized as disposable health and environmental-friendly micro-chips. Using stereo lithography and soft lithography, micro-chambers and micro-channels features have been replicated on zein films and enclosed zein microfluidic devices are created by bonding to glass substrate using a simple vapor-deposition method. The bonding strength of the zein microfluidic devices has been found to exceed the tensile strength of the zein film and hydraulic pressure, and fluid flow through large-area complex microfluidic designs shows no leakage or distortion. High optical clarity and fluorescent imaging in the zein microfluidic devices are demonstrated by visualizing micro-particles and Rhodamine B. Zein microfluidic devices enable truly disposable microfluidics with intrinsic biocompatibility and biodegradability that can be fabricated using existing techniques.

Formation of zein microphases in ethanol-water.

Zein, a major protein of corn, is soluble in binary mixtures of ethanol and water. It has an amphiphilic character and is capable of self-assembly into nano- and microsized rods, spheres, and films upon solvent evaporation. The formation of microspheres is of particular interest for the development of delivery systems. Control over structure formation requires a better understanding of zein behavior in solution. The objective of this work was to investigate the effect of zein concentration and the effect of ethanol-water ratio on the microphase behavior of zein solutions, believed to govern the morphology of microstructures after solvent evaporation. The Flory-Huggins solution theory was applied to model boundary lines between microphases in solution. The study generated information on the zein concentration-ethanol/water ratio conditions where microspheres are formed and provided insight into the microphase behavior of zein ethanolic solutions.

Preparation of zein nanoparticles by pH controlled nanoprecipitation.

Zein is a hydrophobic water insoluble plant protein and the main goal of the present study was to prepare zein nanoparticles using pH controlled nanoprecipitation method. Nanoparticle characteristics such as size, polydispersity index (PI), zeta potential, and encapsulation efficiency were studied using 6, 7-dihydroxycoumarin, as a model hydrophobic compound. Lecithin and pluronic F68 were used as stabilizers. The blank zein nanoparticles had a mean particle size of 460 nm; while coumarin loaded zein nanoparticles had a mean particle size of 365 nm. The encapsulation efficiency of coumarin loaded zein nanoparticles was 62%. The release of coumarin from zein nanoparticles was sustained in phosphate buffer (pH 7.4) for upto 9 days. Overall the results from this study demonstrate a new method for preparing drug loaded zein nanoparticles.

Physical performance of biodegradable films intended for antimicrobial food packaging.

Antimicrobial films were prepared by including enterocins to alginate, polyvinyl alcohol (PVOH), and zein films. The physical performance of the films was assessed by measuring color, microstructure (SEM), water vapor permeability (WVP), and tensile properties. All studied biopolymers showed poor WVP and limited tensile properties. PVOH showed the best performance exhibiting the lowest WVP values, higher tensile properties, and flexibility among studied biopolymers. SEM of antimicrobial films showed increased presence of voids and pores as a consequence of enterocin addition. However, changes in microstructure did not disturb WVP of films. Moreover, enterocin-containing films showed slight improvement compared to control films. Addition of enterocins to PVOH films had a plasticizing effect, by reducing its tensile strength and increasing the strain at break. The presence of enterocins had an important effect on tensile properties of zein films by significantly reducing its brittleness. Addition of enterocins, thus, proved not to disturb the physical performance of studied biopolymers. Development of new antimicrobial biodegradable packaging materials may contribute to improving food safety while reducing environmental impact derived from packaging waste. Practical Application: Development of new antimicrobial biodegradable packaging materials may contribute to improving food safety while reducing environmental impact derived from packaging waste.

Electrospun zein fibers as carriers to stabilize (-)-epigallocatechin gallate.

In this study, a method was developed for continuous electrospinning of ultrafine corn zein protein fibers with diameters ranging from 150 to 600 nm. Fiber-forming solutions with various zein concentrations (10% to 30%, w/w) and aqueous ethanol concentrations (60% to 90%, w/w) were electrospun at 15 and 20 kV. Scanning electron microscopy results showed that the morphology of zein fibers was affected by aqueous ethanol concentration, zein concentration, and the applied voltage. The optimal condition for forming bead-less fibers was found to be 20% protein, 70% alcohol, and 15 kV. The zein fibers resisted solubilization in water, although swelling and plasticization were apparent after the water treatment. The efficacy of zein fibers was tested for stabilization of a green tea polyphenol, (-)-epigallocatechin gallate (EGCG), by incorporating the EGCG in zein fiber-forming solutions. Freshly spun fibers were less effective at immobilizing the EGCG upon immersion in water (82% recovery) as compared to fibers that were aged at 0% relative humidity for at least 1 d (>98% recovery) before water immersion. Fourier transform infrared spectroscopy studies demonstrated that hydrogen bonding, hydrophobic interactions, and physical encapsulation are the major contributors to the stabilization of EGCG in zein fibers in water.

Effect of water vapor on the surface characteristics and cell compatibility of zein films.

In this study, zein microsphere films were fabricated via a solution casting method, and their surfaces were subsequently treated with a higher relative humidity. Changes in surface properties were characterized by contact-angle measurement and scanning electron microscopy (SEM). Human hepatoma cells (BEL-7402) were used as model cells to evaluate cell adhesion, spreading and proliferation on zein films before and after treatment. Additionally, the adhesion and morphologies of rat platelets on zein films were also investigated to evaluate the blood compatibility of the films. The results showed that the hydrophilicity of zein films was changed after treatment, and the surface morphology varied, gradually becoming more smooth and the microspheres in the films disappeared. As the result, the adhesion and proliferation of BEL-7402 cells were significantly improved, while HUVECs were more sensitive than BEL-7402 cells to cell-substrate interactions. Adhesion of rat platelets could be inhibited on the treated zein films. The results suggest that the synergistic actions of hydrophilic properties and micro morphologies are responsible for the cell behaviors.

Topography and biocompatibility of patterned hydrophobic/hydrophilic zein layers.

The topography and biocompatibility of zein layers adsorbed on patterned templates containing hydrophilic and hydrophobic regions were investigated. Nanopatterned templates consisting of hydrophilic lines on a hydrophobic background were drawn by dip-pen nanolithography (DPN) on gold-coated surfaces. 16-Mercaptohexadecanoic acid (COOH(CH(2))(15)SH, MHA) was used as primary ink to generate hydrophilic lines. Unpatterned surfaces were backfilled with 18-octadecanethiol (CH(3)(CH(2))(17)SH, ODT), which generated hydrophobic regions. Zein was allowed to adsorb on patterned surfaces from alcohol-water solutions. The topography of zein deposits was observed by atomic force microscopy (AFM). Height profiles from AFM measurements revealed that zein deposits followed closely the nanopatterned templates. The biocompatibility of zein layers assembled over hydrophilic/hydrophobic micropatterned templates was investigated. Templates containing MHA lines and ODT regions were generated by micro-contact printing (microCP). Mouse fibroblasts seeded on patterned zein layers proliferated on zein deposited over MHA lines, but not on zein over ODT. The experiment indicated that fibroblast cells were able to respond to variations in the underlying surface chemistry, transmitted by the different orientation adopted by zein on the different substrates. This property may be useful in controlling the spatial distribution of cells on patterned protein layers.

The maize floury1 gene encodes a novel endoplasmic reticulum protein involved in zein protein body formation.

The maize (Zea mays) floury1 (fl1) mutant was first reported almost 100 years ago, but its molecular identity has remained unknown. We report the cloning of Fl1, which encodes a novel zein protein body membrane protein with three predicted transmembrane domains and a C-terminal plant-specific domain of unknown function (DUF593). In wild-type endosperm, the FL1 protein accumulates at a high level during the period of zein synthesis and protein body development and declines to a low level at kernel maturity. Immunogold labeling showed that FL1 resides in the endoplasmic reticulum surrounding the protein body. Zein protein bodies in fl1 mutants are of normal size, shape, and abundance. However, mutant protein bodies ectopically accumulate 22-kD alpha-zeins in the gamma-zein-rich periphery and center of the core, rather than their normal discrete location in a ring at outer edge of the core. The 19-kD alpha-zein is uniformly distributed throughout the core in wild-type protein bodies, and this distribution is unaffected in fl1 mutants. Pairwise yeast two-hybrid experiments showed that FL1 DUF593 interacts with the 22-kD alpha-zein. Results of these studies suggest that FL1 participates in protein body formation by facilitating the localization of 22-kD alpha-zein and that this is essential for the formation of vitreous endosperm.

In vivo biocompatibility and mechanical properties of porous zein scaffolds.

In our previous study, a three-dimensional zein porous scaffold with a compressive Young's modulus of up to 86.6+/-19.9 MPa and a compressive strength of up to 11.8+/-1.7 MPa was prepared, and was suitable for culture of mesenchymal stem cells (MSCs) in vitro. In this study, we examined its tissue compatibility in a rabbit subcutaneous implantation model; histological analysis revealed a good tissue response and degradability. To improve its mechanical property (especially the brittleness), the scaffolds were prepared using the club-shaped mannitol as the porogen, and stearic acid or oleic acid was added. The scaffolds obtained had an interconnected tubular pore structure, 100-380 microm in pore size, and about 80% porosity. The maximum values of the compressive strength and modulus, the tensile strength and modulus, and the flexural strength and modulus were obtained at the lowest porosity, reaching 51.81+/-8.70 and 563.8+/-23.4 MPa; 3.91+/-0.86 and 751.63+/-58.85 MPa; and 17.71+/-3.02 and 514.39+/-19.02 MPa, respectively. Addition of 15% stearic acid or 20% oleic acid did not affect the proliferation and osteogenic differentiation of MSCs, and a successful improvement of mechanical properties, especially the brittleness of the zein scaffold could be achieved.

Simple coacervates of zein to encapsulate Gitoxin.

This work reports the use of simple coacervates of the hydrophobic protein zein to encapsulate Gitoxin, a cardiotonic glycoside. The microspheres obtained using ethanol, methanol, iso-propyl alcohol were characterized using viscosity index, scanning electron microscopy (SEM) and laser light scattering particle analyzer. Scanning electron micrographs indicated that the zein film was made of microspheres with diameter in the 1-1.5 microm range, which could be controlled. Sizes of Gitoxin-loaded zein microspheres changed little before and after release of the drug because of conglutination among zein microspheres. Release of Gitoxin from zein microspheres, were performed in vitro to investigate the mechanism of model drug release. The results show that the zein microspheres obtained using ethanol are best suited for use as a sustained-release form of Gitoxin. The microspheres may also be useful in drug targeting system since the diameter of the microspheres is appropriate for phagocytosis by macrophages. Both zein film and Gitoxin-loaded zein microsphere film were effective in suppressing platelet adhesion.

Mechanical properties and in vitro biocompatibility of porous zein scaffolds.

A porous scaffold utilizing hydrophobic protein zein was prepared by the salt-leaching method for tissue engineering. The scaffolds possessed a total porosity of 75.3-79.0%, compressive Young's modulus of (28.2+/-6.7)MPa-(86.6+/-19.9)MPa and compressive strength of (2.5+/-1.2)MPa-(11.8+/-1.7)MPa, the percentage degradation of 36% using collagenase and 89% using pepsin during 14 days incubation in vitro. The morphology of pores located on the surface and within the porous scaffolds showed good pore interconnectivity by scanning electron microscopy (SEM). Rat mesebchymal stem cells (MSCs) could adhere, grow, proliferate and differentiate toward osteoblasts on porous zein scaffold. With the action of dexamethasone, the cells showed a relative higher activity of alkaline phosphatase (ALP) and a higher proliferating activity (p<0.05) than those of MSCs without dexamethasone.