Isolation and Characterization of Zein from Corn Distillers' Grains and Related Fractions

Corn distillers' grains with solubles (CDGS), the major coproduct of fermentation of corn to produce ethanol, were extracted with 0.1M NaOH, 0.1% dithiothreitol (DTT), and 0.5% SDS yielding 35% of the total nitrogen and ≈25% of the protein nitrogen. Gel electrophoresis revealed that the extractable proteins contained zein plus other proteins similar to the extractable proteins from corn flour. Although difficult to extract, the proteins isolated from the fermentation coproducts appeared undegraded and apparently survived gelatinization, fermentation, distillation, and drying during the production of ethanol. Extraction of CDGS with 60% ethanol at 60°C yielded 1.5–3.9% of crude zein. When the ethanol contained DTT, yields of crude zein were increased to 3.2–6.6%. Protein contents of the crude zeins were only 37–57%, indicating that lipids and pigments were coextracted with the ethanol. Gel electrophoresis showed that the protein fractions extracted by ethanol contained primarily α-zein whereas the proteins extracted by ethanol + DTT contained α- + β-zein. Further confirmation of the presence of zein in the crude prolamin preparations was obtained by amino acid analyses. The amino acid compositions of the crude zeins paralleled those of commercial zein and α-zein.     

Solvent Extraction of Zein from Dry‐Milled Corn

Batch extraction of zein from dry‐milled whole corn with ethanol was optimum with 70% ethanol in water, an extraction time of 30–40 min, and temperature of 50°C. High yields (60% of the zein in corn) and high zein contents in the extracted solids (50%) were obtained at a solvent‐to‐solids ratio of 8 mL of 70% ethanol/g of corn. However, zein concentration in the extract was higher at lower ratios. Multiple extraction of the same corn with fresh ethanol resulted in a yield of 85% after four extractions, whereas multiple extractions of fresh corn with the same ethanol resulted in high (15 g/L) zein concentration in the extract. Optimum conditions for batch extraction of zein were 45°C, with 68% ethanol at a solvent‐to‐solids ratio of 7.8 mL/g for an extraction time of 55 min. Column extractions were also best at 50°C and 70% ethanol; a solvent ratio of 1 mL/g resulted in high zein concentrations in the extract (17 g/L) but yields were low (20%).     

Plasticizers for Zein: Their Effect on Tensile Properties and Water Absorption of Zein Films

Cast zein films are brittle at room conditions, so plasticizers are added to make them more flexible. The tensile properties of these films are known to be affected by the relative humidity (RH) of the ambient air. However, little is known about how the plasticizers are affected by RH. Cast zein films were plasticized with either glycerol (GLY), triethylene glycol (TEG), dibutyl tartrate (DBT), levulinic acid (LA), polyethylene glycol 300 (PEG), or oleic acid (OA). Mechanical properties and moisture content (MC) of the films were measured after one week of storage at 3, 20, 50, 70, 81, and 93% RH. The relative humidity of the films' storage had a great effect on the films' tensile properties. All the films' tensile strength and Young's modulus values decreased as RH increased. Films containing DBT, TEG, LA, or PEG showed an increase in the percent elongation with increasing RH. Films containing GLY, OA, or no plasticizer did not show any increase in percent elongation as RH increased. The changes seen in tensile properties with increasing RH are because of zein's hygroscopic nature. The absorbed water will further plasticize the zein. The type of plasticizer used determined the extent of the changes seen in the tensile properties of films stored at different RH values. Depending on the plasticizers used in the film, there were large differences in the amount of water absorbed. Films increasingly absorbed water depending on the plasticizer they contained in the order GLY > TEG > LA > PEG > NONE > DBT > OA. Films containing hygroscopic plasticizers like TEG absorbed too much water at high RH and became weak, but they absorbed enough water at lower RH values to not be brittle. While films containing the more hydrophobic plasticizer DBT were brittle at intermediate RH values, they had good mechanical properties at high RH values.     

Factors Affecting Yield and Composition of Zein Extracted from Commercial Corn Gluten Meal

Twelve corn gluten meal samples obtained from six wet-milling plants were processed into zein. Zein was extracted using 88% aqueous isopropyl alcohol at pH 12.5, followed by chilling. Protein recovery ranged from 21.3 to 32.0%, and protein purity ranged from 82.1 to 87.6%. Protein recovery increased as the protein purity increased (r = 0.76) (P < 0.01). One of the major factors influencing extraction yield was protein composition; especially α-zein content, which ranged from 53.4 to 64% of the total protein in the corn gluten meal samples. The intensity of red color of the corn gluten meal was negatively correlated with protein recovery and zein purity (r = -0.66 and -0.72, respectively) (P < 0.02).     

Cast Films from Soy Protein Isolates and Fractions

Glycerol-plasticized soy protein films were cast from alkaline aqueous film-forming solutions of laboratory-prepared 7S, 11S, and soy isolate (LSI) fractions and from commercial soy isolate (CSI). Tensile strength (TS), elongation at break (E), water vapor permeability (WVP), total soluble matter (TSM), protein solubility (PS), and Hunter L, a, and b color values of these films were determined. The 11S films had greater TS than 7S films (P < 0.05), while LSI films had greater TS than CSI films (P < 0.05). No significant differences were detected among mean E values and among mean WVP values of all films (P > 0.05). The 7S films had higher TSM and PS values than 11S films (P < 0.05). CSI films were significantly darker (lower L value) and more yellow (greater positive b value) than LSI films (P < 0.05).     

Properties and Microstructure of Plasticized Zein Films

A new method for preparation of zein films involving plasticization of zein with oleic acid to form an intermediate moldable resin was presented. The resin was stretched over rigid frames to form thin membranes that were set in flexible films. The objective of the study was to investigate the effect of film preparation method on film properties. Tensile properties, microstructure, and thermal behavior of zein films plasticized with oleic acid were investigated for films prepared by conventional casting from ethanol solutions and by stretching of plasticized resins. Cast films were stiff and brittle, whereas resin films showed more flexibility and toughness. Differential scanning calorimetry thermograms of cast films indicated phase separations were generated when heated that were not observed for resin films. Microstructure images showed a higher degree of structure development and orientation in resin than in cast films. Glass-transition temperatures of resin films were measured at -94 and 104.4° C, indicating the film remained flexible through a wide temperature range. Resin film flexibility and toughness were attributed to effective plasticization that led to fiber formation and orientation.     

Zein: A History of Processing and Use

Corn is the largest and most important agricultural commodity in America. Zein, one of the components in corn, has long been investigated for uses other than food and feed. Zein is a unique and complex material, and it is one of the few cereal proteins extracted in a relatively pure form. Today, because of environmental concerns, interest in zein utilization is again growing. Some of the more important research on zein is more than 50 years old. Most of this work has been either forgotten, lost, or difficult to locate. Much of this work was done at the USDA laboratory in Peoria, IL. Since most early zein literature is still easily accessible at that laboratory, this review on zein has been prepared making use of this old literature. This review reexamines the old literature and reconciles it with new zein research to illustrate some of the unique properties of and opportunities for zein.     

Effects of Extrusion and Starch Removal Pretreatment on Zein Proteins Extracted from Corn Gluten Meal

In this study, the structure and selected properties of zeins extracted from corn gluten meal (CGM) pretreated by extrusion and removal of starch were investigated. The structure and properties of the zeins from pretreated CGM changed significantly. Pretreatments can decrease the extraction yields of zeins and change the granule shape and size of zein aggregates. The studies indicated that extrusion and removal of starch can significantly decrease the thermal enthalpy (ΔH₁ and ΔH₂) of zein from 1.94 ± 0.20 to 0.19 ± 0.10 and from 107.20 ± 0.80 to 78.62 ± 2.30 and J/g, respectively. The SDS‐PAGE results confirmed that the molecular weight of zeins from CGM was 24,000 and 27,000, and the molecular weight of zeins did not change with the pretreatment. On the other hand, the circular dichroism spectroscopy results showed that the processing of extrusion and removal of starch can change the secondary structure content of β‐sheets and β‐turns; these results indicated that extrusion and removal of starch can significantly break the secondary structure of zeins. Furthermore, extrusion and removal of starch can change the sulfhydryl content of zeins. The obtained results provided some fundamental information that is useful for further modification of CGM to improve its functional properties and industrial applications.     

Improved Isolation of Zein from Corn Gluten Meal Using Acetic Acid and Isolate Characterization as Solvent

An improved means of isolating zein is needed to develop new uses for corn zein. We have measured the yield of zein and evaluated the ability of acetic acid to remove zein from corn gluten meal, distillers dried grains, and ground corn using acetic acid as solvent. Acetic acid removed zein more quickly, at lower temperatures, and in higher yields when compared with alcoholic solvents. After 60 min at 25°C, ≈50% of the zein in corn gluten meal was removed. A step change in yield from 43 to 50% occurs as the extraction temperature is increased from 40 to 55°C after mixing for 30 min at 25% solids. The protein composition of the zein removed from corn gluten meal using acetic acid is very similar to that of commercial zein by SDS‐PAGE. The zein obtained from corn gluten meal using acetic acid had higher amounts of fatty acids and esters according to IR analysis, leading to slightly lower protein content. Films made from zein extracted from corn gluten meal using acetic acid had lower tensile strength (≈60% lower) than films produced from commercial zein. Fibers with very small diameter (0.4–1.6 μm) can be produced by electrospinning using the AcOH solution obtained after corn gluten meal extraction.     

Effect of endogenous triacylglycerol hydrolysates on the mechanical properties of Zein films from ground corn

Dry-milled yellow corn and freshly ground food and nonfood grade yellow and white hybrid corn kernels were pretreated in a solution of lactic acid and sodium metabisulfite followed by extraction with 70% ethanol. Zein was precipitated from the extract by reducing the ethanol content of the extract to 40%. Lipid associated with the zein isolates was between 15 and 20% and contained mostly endogenous free fatty acids. The effect of the endogenous free fatty acids on zein isolate films, with and without free fatty acids, was determined by measuring various film properties. Stress-strain measurements indicated 40-200% greater elongation for zein films containing endogenous free fatty acids. Films prepared from zein isolated from preground corn stored for approximately 4 months (27 degrees C, 17% relative humidity) had approximately 3 times greater elongation values than zein films prepared from freshly ground corn.     

Properties and Microstructure of Zein Sheets Plasticized with Palmitic and Stearic Acids

Interest in biodegradable materials for packaging and agricultural uses has grown in recent years. Plant proteins have been proposed as inexpensive, renewable, and abundant feedstock. Corn zein was investigated based on value-added considerations and on the unique thermoplastic and hydrophobic properties of zein. Films prepared from zein are known to be tough and resistant, but also hard and brittle, thus requiring the addition of plasticizers to improve flexibility. The objectives of this research were to study the tensile properties, water absorption, and microstructure of zein sheets plasticized with palmitic and stearic acids. Both palmitic and stearic acids showed similar effects as plasticizers of zein. Tensile strength of zein sheets increased with the addition of low levels of plasticizers. However, beyond a critical point, tensile strength decreased with further addition of fatty acids. Water absorption decreased continuously with increasing fatty acid content. Kinetic parameters indicated fatty acids decreased water absorption by decreasing the saturation level of zein sheets. Coating zein with flax oil decreased the rate of water absorption by sealing off surface pores. Scanning electron micrographs of zein sheets showed the development of layered structures as fatty acid content increased. Zein-fatty acid layers were believed to be responsible for the increased tensile strength of plasticized zein sheets and to have contributed to increased resistance to water absorption.     

Zein Compositions of Mechanically Separated Coarse and Fine Portions of Maize Kernels

Coarse and fine kernel portions from 24 maize inbreds (six grown in two years) and four hybrids were separated by grinding and sifting. Zeins from both portions of all genotypes were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Results agreed, in general, with those of a previous study in which endosperm separation was done manually, suggesting that endosperm fractionation can be done by mechanical means. Compositions of zeins, as revealed by RP-HPLC and SDS-PAGE, support the hypothesis that zeins help determine maize endosperm hardness.     

Isolation and Characterization of Cotyledon Fibers from Peas,Lentils, and Chickpeas

Methods were developed to efficiently isolate legume cotyledon fibers with relatively high yields and purities. Seeds of pea (Pisum sativum), chickpea (Cicer arientinum), and lentil (Lens culinaris) were roller milled into flour and fractionated into prime starch, tailings starch, and water solubles. Insoluble dietary fiber was isolated from tailings starch by wet screening on sieves with openings ranging from 53 to 90 μm. Yield of insoluble fiber using a sieve with 53‐μm openings ranged from 49.7 to 59.2% of insoluble fiber in flour with purities ranging from 85.5 to 87.3%. Soluble dietary fiber was isolated from the water‐soluble fraction following acid precipitation of soluble protein at pH 4. Soluble fiber yield ranged from 83.3 to 89.6% of flour soluble fiber with purities ranging from 64.5 to 70.6%. Glucose was the most common sugar component of hulls and soluble cotyledon fibers, while arabinose was the main sugar in insoluble fibers. Insoluble fiber exhibited significantly higher swelling capacities and water and oil binding capacities in comparison to hulls and soluble cotyledon fibers. Apparent viscosities of soluble cotyledon fibers ranged from 3.13 to 3.43 Pa•sec and exhibited Newtonian characteristics.     

REVIEW: Zein Extraction from Corn,Corn Products,and Coproducts and Modifications for Various Applications: A Review

Corn can be fractioned to produce starch, fiber, oil, and protein in relatively pure forms. The corn kernel contains 9–12% protein, but half of this is an industrially useful protein called zein. Dry milled corn (DMC), corn gluten meal (CGM), and distiller's dried grains with solubles (DDGS) are all coproducts from corn that contain zein and are used for zein extraction. Because it is insoluble in water, zein has found uses in many products such as coatings, plastics, textiles, and adhesives. Newer applications are taking advantage of zein's biological properties for supporting growing cells, delivering drugs, producing degradable sutures, and producing biodegradable plastics. This review covers zein characteristics and nomenclature, past and current practices in processing and extraction of zein from corn products and coproducts, and the modifications of zein for various applications.     

Improvement of Sorghum-Wheat Composite Dough Rheological Properties and Breadmaking Quality Through Zein Addition

Addition of sorghum flour to wheat flour produces marked negative effects on rheological properties of dough and loaf volume. Although there are notable differences in the chemical composition of sorghum proteins (kafirins) compared with wheat gluten that might imply poor functionality in breadmaking systems, a larger constraint may be the unavailability of kafirins due to encapsulation in protein bodies. In this study, zein, the analogous maize prolamin to kafirin, was used to determine the potential effects of protein-body-free prolamins on dough rheology and baking quality of wheat-sorghum composite flour. Mixograms run at 35°C (above the glass transition temperature of zein) were significantly (P < 0.01) improved with addition of zein. Mixogram peak heights increased while mixing time decreased uniformly with addition of zein. Dough extensibility studies showed an increase in maximum tensile stress, while baking studies showed an increase in loaf volume with increasing amounts of added zein. These data are supported by a previous study showing that, in a model system, zein mixed with starch can form viscoelastic networks, and suggest that kafirin, if made available, could contribute to dough formation.     

Effect of Solvent and Temperature on Secondary and Tertiary Structure of Zein by Circular Dichroism

Circular dichroism studies were performed on zein to determine how the secondary and tertiary structure changes with different solvents, temperatures, or pH. Alcoholic solvent type and common denaturants such as SDS and low amounts of urea had little effect on the secondary structure of zein. Utilization of dimethylformamide or acetic acid as solvent gave changes in tertiary structure. Solutions of zein in 8M urea produced solutions with large changes in tertiary structure. The dissolution of zein in 50 mM sodium hydroxide produces a zein with large changes in secondary and tertiary structure and little loss in primary structure. Increasing the temperature of zein to 70°C in 80% ethanol-water gave reversible changes in the primary structure (20% reduction in absolute magnitude of [θ]λ at 208 and 222 nm) and tertiary structure (40% reduction in absolute magnitude of [θ]λ at 268 nm).     

Photocatalytic Degradation of Textile Dye and Wastewater

In this study, the photocatalytic degradation of commercial azo dye (Remazol Red 133) in the presence of titanium dioxide (TiO₂) suspensions as photocatalyst was investigated. The effect of various operational parameters, such as pH of dye solution and catalyst concentration on the photocatalytic degradation process, was examined. The mineralization of dye was also evaluated by measuring the chemical oxygen demand of the dye solutions. The extent of photocatalytic degradation was found to increase with increasing TiO₂ concentration. For the Remazol Red dye solutions, a 120-min treatment resulted in 97.9% decolorization and 87.6% degradation at catalyst loading of 3 g/L. Experiments using real textile wastewater were also carried out. Textile wastewater degradation was enhanced at acidic conditions. The decolorization and degradation efficiencies for textile wastewater were 97.8% and 84.9% at pH 3.0, catalyst loading of 3 g/L, and treatment time of 120 min.     

Developments in the Science of Zein,Kafirin, and Gluten Protein Bioplastic Materials

Despite much research, there are very few commercial prolamin bioplastics. The major reason, apart from their high cost, is that they have inferior functional properties compared with synthetic polymer plastics. The inferior functional properties are because the prolamins are complex, each consisting of several classes and subclasses, and the functional properties of their bioplastics are greatly affected by water. Prolamin bioplastics are produced by controlled protein aggregation from a solvent or by thermoplastic processing. Recent research indicates that aggregation occurs by polypeptide self‐assembly into nanostructures. Protein secondary structure in terms of α‐helical and β‐sheet structure seems to play a key but incompletely understood role in assembly. Also, there is inadequate knowledge as to how these nanostructures further assemble and organize into the various forms of prolamin bioplastics such as films, fibers, microparticles, and scaffolds. Many methods have been investigated to improve prolamin bioplastic functionality, including better solvation of the prolamins, plasticization, physical and chemical cross‐linking, derivatization, and blending with synthetic and natural polymers, and some success has been achieved. The most promising area of commercialization is the biomedical field, in which the relative hydrophilicity, compatibility, and biodegradability of, particularly, zein and kafirin are advantageous. With regard to biomedical applications, “supramolecular design” of prolamin bioplastics through control over inter‐ and intramolecular weak interactions and disulfide/sulfhydryl interchange appears to have considerable potential.