Nanoscale characterization of zein self-assembly.

Zein, a major protein of corn, is rich in α-helical structure. It has an amphiphilic character and is capable of self-assembly. Zein can self-assemble into various mesostructures that may find applications in food, agricultural, and biomedical engineering. Understanding the mechanism of zein self-assembly at the nanoscale is important for further development of zein structures. In this work, high-resolution transmission electron microscopy (TEM) images revealed nanosize zein stripes, rings, and discs containing a 0.35 nm periodicity, which is characteristic of ß-sheet. TEM images were interpreted in terms of the transformation of original α-helices into ß-sheet conformation after evaporation-induced self-assembly (EISA). The presence of ß-sheet was also detected by circular dichroism (CD) spectroscopy. Zein ß-sheets self-assembled into stripes, which curled into rings. Rings formed discs and eventually spheres. The formation of zein nanostructures was believed to be the result of ß-sheet orientation, alignment, and packing.

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.

Formation and Characterization of Zein-Based Oleogels.

Oleogels are interesting as a result of their ability to hold large amounts of oil in a semi-solid gel structure. In the food industry, oleogels are most often investigated as substitutes for saturated and trans fats. In this work, the lyotropic formation of ethanol/zein/oleic acid gels was observed qualitatively and ternary phase diagrams were constructed to map the observations. The viscoelastic parameters G' and G'' were measured to confirm gel formation as observed in phase diagrams. Ultrasmall X-ray scattering was used to study the microstructural organization of ethanol/zein/oleic acid gels. Data suggested that the primary unit or building block for gel network structures was the rod-shaped zein molecule. Ultrasmall X-ray data suggested that zein/oleic acid gels have a highly organized microstructure, possibly the result of zein self-assembly. Zein was considered an effective oleogelator in ethanol/zein/oleic acid systems.

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.

Fourier transform infrared spectra of drying oils treated by irradiation.

Drying oils, such as linseed oil and tung oil, have the potential as coating materials to improve barrier properties of biobased packaging films. Oil drying is a chemical reaction in which polyunsaturated fatty acids undergo autoxidation. During drying, oils polymerize and form water-resistant films. However, drying rates tend to be too slow for practical applications. Metal driers are used in the paint industry to accelerate drying, but often driers are not safe for food contact. The objective of this work was to investigate the effect of ionizing radiation on the oxidation or drying rate of drying oils. The effect of irradiation dose on the drying rate of linseed and tung oils was monitored by FTIR spectroscopy. The peak at 3010 cm (-1) was found to be a useful index of oxidation rate. The decrease in peak intensity with time was fitted with exponential functions of the form Abs = Abs 0 exp (- t/ k), where Abs 0 is the initial absorbance and 1/ k is the rate constant for the oxidation process. Values for k were 9.91 ( R (2) = 0.98), 6.59 ( R (2) = 0.95)n and 6.44 ( R (2) = 0.97) for radiation levels of 0, 50, and 100 kGy, respectively. The k values suggested that the oxidation rate increased as the radiation dose increased from 0 to 50 kGy. A further increase to 100 kGy had only a limited effect.

Zein dynamic adsorption to carboxylic and alkyl coated surfaces.

The adsorption of a commercial zein sample on carboxylic (COOH) and alkyl (CH(3)) surfaces was monitored by high time resolution surface plasmon resonance. Zein showed higher affinity and higher mass adsorption on carboxylic than alkyl surfaces. A zein layer specific for each surface was obtained after flushing off loosely bound zein with 75% 2-propanol solutions. Zein deposits were examined under atomic force microscopy. Differences in layer thickness between carboxylic and alkyl surfaces were explained in terms of zein adsorption footprint.

Cell spreading and viability on zein films may be facilitated by transglutaminase.

Zein is a biocompatible corn protein potentially useful in the development of biomaterials. In this study, the deposition of zein on oxygen plasma treated glass cover slips significantly enhanced cell spreading and viability. The mechanism for cellular response to zein coated surfaces was thought to involve the polyglutamine peptides on the zein structure. We hypothesized that zein was a substrate for tissue transglutaminase (tTG), an extracellular enzyme involved in cell-surface interactions. SDS-PAGE results suggested an interaction between zein and tTG, where zein was the glutamine donor. Cross-linking between zein and tTG may be the first step in successful cell adhesion and spreading.

Properties of zein films coated with drying oils.

Zein films prepared by resin were coated with either flax oil or tung oil and cured by UV- or gamma-radiation. Coated zein films were then evaluated for tensile and water barrier properties. Film microstructure was examined by transmitted light microscope. Tensile strength, elongation, and toughness of oil-coated samples increased substantially with respect to uncoated films. Flax oil coated samples showed an increase in elongation of 300%. It was suggested that oil coatings fill in pinholes and cracks existing in zein films, affecting their mechanical properties. Water vapor permeability also decreased statistically for coated film (except for tung oil coated-UV treated films), suggesting water vapor transfer was controlled by film hydrophobicity and microstructure. Moreover, the liquid water transmission rate of coated films was at least 10 times slower than for control films. Examination of film microstructure revealed that flax oil coatings had uniform coverage and smooth finish, which explained their high elongation, low water vapor permeability. Tung oil coatings cured under UV light showed patterns of intertwined dark and light regions, which may be caused by cross-linking and drying at different times. The oriented structures were found when tung oil coatings were cured by gamma-radiation.

Effects of processing on the structure of zein/oleic Acid films investigated by x-ray diffraction.

Zein films plasticized with oleic acid were formed by solution casting, by the stretching of moldable resins, and by blown film extrusion. The effects of the forming process on film structure were investigated by X-ray diffraction. Wide-angle X-ray scattering (WAXS) patterns showed d-spacings at 4.5 and 10 A, which were attributed to the zein alpha-helix backbone and inter-helix packing, respectively. The 4.5 A d-spacing remained stable under processing while the 10 A d-spacing varied with processing treatment. Small-angle X-ray scattering (SAXS) detected a long-range periodicity for the formed films but not for unprocessed zein, which suggests that the forming process-promoted film structure development is possibly aided by oleic acid. The SAXS d-spacing varied among the samples (130-238 A) according to zein origin and film-forming method. X-ray scattering data suggest that the zein molecular structure resists processing but the zein supramolecular arrangements in the formed films are dependent on processing methods. Structural model for a zein molecular aggregate (based on Matsushima et al.10). Rectangular prisms of individual zein molecules are hexagonally aligned parallel to each other.

Water sorption properties of extruded zein films.

Water interactions in extruded zein films were investigated through moisture sorption isotherms. Sorption isotherms of zein products were affected by composition and structure morphology. Zein powder showed moisture sorption hysteresis, which was not observed in extruded samples. Extruded samples held less moisture than zein powder, while films containing oleic acid showed further reduction in moisture uptake. Brunauer, Emmett, and Teller (BET) and Guggenheim, Anderson, and De Boer (GAB) models fitted well the moisture sorption isotherms of zein products. Monolayer values estimated by BET and GAB models were consistent with predictions based on zein structural models. Water vapor permeability (WVP) of zein films was affected by the relative humidity of testing environment. Higher relative humidity resulted in higher WVP.

Protein-lipid interactions in zein films investigated by surface plasmon resonance.

Experiments on the adsorption of alpha-zein (characterized by SDS-PAGE) from aqueous ethanol and 2-propanol solutions onto hydrophilic and hydrophobic surfaces are reported. Zein adsorption onto self-assembled monolayers (SAMs) was detected by surface plasmon resonance (SPR). Gold substrates were prepared by thermal evaporation on glass slides. Gold-coated surfaces were modified by depositing SAMs of either a long-chain carboxylic acid terminated thiol [COOH(CH2)(10)SH] or a methyl-terminated alkanethiol [CH3(CH2)(7)SH]. Experimental measurements indicated that zein interacted with both hydrophilic and hydrophobic surfaces. Zein concentration affected the thickness of bound zein layers. The estimated thickness of the zein monolayer deposited on hydrophilic surfaces was 4.7 nm. Zein monolayer thickness on hydrophobic surfaces was estimated at 4.6 nm. The topography of zein layers was examined by atomic force microscopy (AFM) after solvent was evaporated. Surface features of zein deposits depended on the adsorbing surface. On hydrophilic surfaces, roughness values were high and distinct ring-shaped structures were observed. On hydrophobic surfaces, zein formed a uniform and featureless coverage.

Analysis of zein by matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) was used to analyze the protein composition of corn prolamine (zein). Mass spectra were obtained from commercial zein and zein extracted with aqueous 2-propanol and aqueous ethanol from consumer corn meal. For the commercial zein, three major zein fractions with m/z 26.8k, 24.1k, and 23.4k were clearly seen with two minor fractions (m/z 14.5k and 20.4k) also present. As compared with the results from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these three fractions were identified as alpha-zeins (24.1k and 23.4k combined as Z19; 26.8k as Z22). When extracted with 55% aqueous 2-propanol, three alpha-zein fractions with m/z 26.8k, 24.1k, and 23.4k were predominant. When extracted with ethanol, extraction temperature had an effect on the final products. When extracted with 75% aqueous ethanol at room temperature, alpha-zein and some 17-18k species were observed, whereas at 60 degrees C, a small amount of delta-zein was also present. Comparison of the MALDI/MS results with SDS-PAGE and gene sequence analysis shows that the MALDI/MS method is superior to SDS-PAGE in having higher resolution and mass accuracy.

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.

Zein adsorption to hydrophilic and hydrophobic surfaces investigated by surface plasmon resonance.

Zein, the prolamine of corn, has been investigated for its potential as an industrial biopolymer. In previous research, zein was plasticized with oleic acid and formed into sheets/films. Physical properties of films were affected by film structure and controlled in turn by zein-oleic acid interactions. The nature of such interactions is not well understood. Thus, protein-fatty acid interactions were investigated in this work by the use of surface plasmon resonance (SPR). Zein adsorption from 75% aqueous 2-propanol solutions, 0.05% to 0.5% w/v, onto hydrophilic and hydrophobic self-assembled monolayers (SAMs) formed by 11-mercaptoundecanoic acid and 1-octanethiol, respectively, was monitored by high time resolution SPR. Initial adsorption rate and ultimate surface coverage increased with bulk protein concentration for both surfaces. The initial slope of plotted adsorption isotherms was higher on 11-mercaptoundecanoic acid than on 1-octanethiol, indicating higher zein affinity for hydrophilic SAMs. Also, maximum adsorption values were higher for zein on hydrophilic than on hydrophobic SAMs. Flushing off loosely bound zein in the SPR cell allowed estimation of apparent monolayer values. Differences in monolayer values for hydrophobic and hydrophilic surfaces were explained in terms of zein adsorption footprint.