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.     

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%).     

Tensile properties and water absorption of zein sheets plasticized with oleic and linoleic acids.

Corn zein has been investigated for fabrication of biodegradable packaging materials. Our objective was to investigate the effect of added plasticizers, oleic and linoleic acids, on tensile properties and water absorption of zein sheets. Moldable resins were precipitated from aqueous ethanol dispersions of zein and fatty acids and rolled into sheets of approximately 0.5 mm in thickness. To increase plasticization effects, zein-oleic acid sheets were replasticized by heating them in fatty acid baths. Plasticization resulted in flexible sheets of high clarity, low modulus, and high elongation and toughness, although low tensile strength. Water absorption of zein sheets was lowered by plasticization, attributed in part to reduced mass fraction of zein. Polymerization of linoleic acid may have sealed off pores on sheet surfaces, thus slowing water absorption.     

Quality of Corn Oil Obtained by Sequential Extraction Processing

Sequential extraction processing (SEP) is a new approach to fractionating dried, flaked corn using 95% ethanol. In the original process, corn oil was extracted at 76°C in a countercurrent mode while simultaneously dehydrating the ethanol. This resulted in 20% of the protein (predominantly zein) coextracting with the oil. The process was modified to reduce the amount of coextracted protein. One modification (mSEP1) was to use a blend of 30% hexane and 70% ethanol at 56°C. A second modification (mSEP2) used a longer extraction column (L/D ratio 15) to replace the column with L/D 2 used in the original SEP system. To determine the effect of the modifications on oil quality, the quality of the crude corn oils produced from the modified SEP processes were compared with the quality of oil from the original SEP. To evaluate the quality of the three crude oils produced by SEP with the process typically used in industry, they were compared with the quality of laboratory hexane‐extracted corn oil. The results of the three SEP oils exhibited larger concentrations of fatty acids, phospholipids, and carotenoids, smaller concentrations of triacylglycerols, and darker red color than the hexane‐extracted oil. The oils from the two modified SEP processes contained smaller concentrations of free fatty acids and phospholipids and larger concentrations of triacylglycerols and carotenoids than the original SEP oil. In spite of the improvements to the oil through process modifications, the mSEP1 and mSEP2 oils exhibit greater refining losses than hexane‐extracted oil.     

Zein Batch Extraction from Dry-Milled Corn: Cereal Disintegration by Dissolving Fluid Shear

Corn particles were extracted in an agitated vessel with a 4:1 mass ratio of 70% ethanol to corn for periods of 1–6 hr at ambient temperature. The extract solution was filtered and centrifuged to remove suspended particles after extraction and then diluted to 40% ethanol to precipitate extracted solute. Measurements of the mass of suspended particles separated by centrifugation indicate that mixing the corn particles with the ethanol dissolves and weakens the protein between cells and between starch granules within cells near the particles' surface. Under the conditions of this study, corn particles release starch granules more rapidly than the protein bodies dissolve, as indicated by analysis of the centrifuged particles. The diffusion coefficient for ethanol solution in corn was estimated and compared with a coefficent derived from a fit of the trend in the rate of release of fine particles from the milled corn. The diffusion coefficient of pure zein in a stagnant 70% ethanol solution was estimated from the measurement of weight loss by a ball of zein. Analysis of the ambient temperature protein extraction rate indicates that 2-mm particles exhibit more convective mass transfer than 20-μm particles.     

Isolation of Zein Using 100% Ethanol

Traditionally, zein is isolated and recovered from corn gluten meal (GCM) using aqueous alcohol as the solvent. Recovery of zein from this solvent is inconvenient and costly. Zein is insoluble in 100% ethanol at room temperature, but it is soluble at 120°C in ethanol. Absolute ethanol effectively extracted zein from CGM, distillers dried grains (DDG), and ground corn. Zein was extracted from CGM with absolute ethanol in a high‐pressure reactor at 130°C. After extracting at 130°C for 45 min, the solution was pumped out of the extractor and allowed to cool. Upon cooling, the zein precipitated from solution. The precipitate was removed from the solution and air‐dried, resulting in 14% recovery of the starting material. The recovered precipitate had an average protein content of >90% on a dry basis, accounting for ≈20% of the CGM protein and recovered ≈35% of its zein. No differences were seen in the amount of zein extracted from CGM samples that were hand‐collected off the dewatering screen and gently dried, versus commercial CGM samples. The commercial CGM did produce a greater amount of solubles. The extraction procedure also worked at temperatures as low as 90°C. The lower temperature did produce lower yields of extracted zein. The zein extracted at the lower temperatures was less brown, but zein extracted at either temperature was almost fully soluble in traditional zein solvents.     

Optimizing Extraction of Zein and Glutelin‐Rich Fraction During Sequential Extraction Processing of Corn

This study was conducted to improve yields and qualities of corn protein co‐products produced by the sequential extraction process (SEP), a process using ethanol to fractionate corn in producing fuel ethanol. A two‐stage extraction protocol was evaluated to recover zein and subsequently recover a glutelin‐rich fraction (GRF). After the simultaneous oil‐extraction and ethanol‐drying step of SEP, zein was extracted from the anhydrous‐ethanol‐defatted, flaked corn by using 70% (v/v) ethanol at 60°C for 1.5 hr in a shaking water bath. Zein was recovered by ultrafiltering and then drying in a vacuum‐oven. Zein yield was 65% of the available zein in the flaked corn. SDS‐PAGE band patterns of the recovered zein closely resembled that of commercial zein. After zein extraction, the GRF was extracted using 45% ethanol and 55% 0.1M NaOH at 55°C for 2 hr. The extract was concentrated by ultrafiltration and then freeze‐dried. GRF yield was ≈65% of the available protein. Freeze‐dried GRF contained 90% crude protein (db), which classified the protein as a protein isolate. As with the protein concentrate from the original SEP, the GRF isolate was highly soluble in water at pH ≥ 7, had good emulsifying and foaming properties, formed stable emulsions, and was heat‐stable.     

Tensile Properties of Extruded Corn Protein Low‐Density Polyethylene Films

The strength of films extruded from powder blends of corn zein or corn gluten meal (CGM) with low‐density polyethylene was investigated. Tensile strength, percent elongation at break, and elastic modulus of the extruded films were measured. The tensile strength decreased from 13 MPa to ≈10.5 MPa with zein addition, while CGM addition resulted in tensile strength of ≈6 MPa. The higher the level of biological material (CGM or zein) in the films the lower the tensile properties. Films containing CGM exhibited significantly lower tensile properties than those containing zein. Extrusion processing of biological films is a step toward commercial viability.     

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.     

Quantitative Analysis of Corn Zeins by Capillary Electrophoresis

Kjeldahl analysis is commonly used to measure zein proteins in corn maize (N × 5.7) with no attempt to eliminate contribution from other nitrogen sources. In this study, dry milled corn was extracted with 70% ethanol or 0.1N NaOH and the zein content of the extract measured using capillary electrophoresis in the presence of sodium dodecyl sulfate. The amount of zein protein in alcohol extracts, using this method, was in good agreement with that determined by Kjeldahl nitrogen analysis. However, less than half of the Kjeldahl nitrogen in the alkaline extracts could be attributed to corn zein. Reproducibility expressed as relative standard deviation for migration time and peak area was 0.10 and 1.05, respectively. The technique permits rapid analysis of a large number of samples without interference from other compounds present in the extracts.     

Sequential Extraction Processing of High‐Oil Corn

The Sequential Extraction Process (SEP), a process that uses ethanol to fractionate corn into high‐value co‐products when producing fuel ethanol, was evaluated using high‐oil corn (HOC). Oil and protein recoveries, ethanol‐drying capability, and oil and protein properties were compared with those produced from normal soft dent corn (SDC) using SEP. Moisture adsorption capacities (≈24 g of water/kg of corn) and oil recoveries (>95%) were nearly identical for both corn types. However, oil yield from HOC (7.1 g of oil/100 g of corn) was 65% more than that of SDC (4.3 g of oil/100 g of corn). HOC crude oil was less red and contained lower free fatty acid and phosphatide contents than did SDC oil. HOC zein contained higher crude protein content (86% db) than did SDC zein (79% db). The freeze‐dried glutelin‐rich fractions (GRF) from both types of corn contained >90% (db) crude protein, which meet the criteria of protein isolates. SEP GRF protein isolates had >85% protein soluble in water at pH ≥ 7. Both GRF protein isolates from HOC and SDC were heat‐stable, had good emulsifying capacities, and produced highly stable emulsions. They also had substantial foaming capacities, but the foam from HOC GRF protein isolate was significantly more stable than the foam from SDC GRF protein isolate. SEP is a suitable process for recovering oil and protein products from HOC corn when producing fuel ethanol and, if competitively priced, HOC is a preferred feedstock to SDC because of higher yields and qualities of valuable co‐products when using HOC.     

Adhesive properties of corn zein formulations on glass surfaces

Adhesive properties on glass of commercial zein and an inexpensive zein-lipid mixture isolated from dry-milled corn were investigated. A method was developed for uniformly preparing bonded glass panels and measuring the amount of pull required to separate the panels. The adhesive strength of commercial zein to glass was greater at 29% than at 52% relative humidity (RH). Bonded samples prepared from zein isolates were less sensitive to changes in RH. Bonds using commercial zein formulations containing plasticizer reached a maximum strength at 10% poly(ethylene glycol) regardless of RH. Formulations that required the least amount of ethanol (35-42%) were obtained by adjusting its pH to 3 or 10 with a volatile acid or base. These formulations completely bonded to the glass panels at low sample concentrations as estimated by 100% cohesive failure and exhibited lower Young's Modulus values than most of the other bonding materials tested. Samples bonded with a polyvinyl acetate emulsion adhesive were not as strong as the zein-bonded samples and were sensitive to changes in RH.     

电场处理改善玉米醇溶蛋白膜理化性质

传统浇铸法制备的玉米醇溶蛋白薄膜表面粗糙,机械性质及耐水性较差。为了改善玉米醇溶蛋白理化性质,在传统浇铸法膜制备过程中引入平行匀强电场(1~5 A/m2)处理蛋白成膜液。经过电场处理后,玉米醇溶蛋白表面光滑、形状完整。试验结果表明:电场处理可改善薄膜力学性质、表面疏水性、水蒸气透过率等性质;随着电流密度的增大,薄膜拉伸强度、断裂伸长率、水蒸气透过率、吸水率以及表面接触角呈现规律性增加或者减小;电场处理下薄膜热特性改变,与未处理组相比变性温度略有提高,最大增幅为19.5℃。当电流密度为4 A/m2时,薄膜理化性质较佳:拉伸强度、断裂伸长率分别为73.09 MPa和9.68%,吸水率降低至14.87%,水蒸气透过率为2.55×10-8 g·m/(m2·h·Pa),静态接触角为62.18°,变性温度提高到118.39℃,热稳定性提高,薄膜表面光滑。电场可诱导成膜液中分子有序性排列,提高薄膜均一性;通过调节电流密度可得到具有一定力学强度和亲/疏水性的薄膜。试验结果为制备具有特定功能性的纯玉米醇溶蛋白薄膜材料提供了理论依据。     

Reducing the brittleness of zein films through chemical modification

Zein protein is a major coproduct of biofuel from corn. To reduce the brittleness of zein films, a new type of zein-based biomaterial, was synthesized by chemical modification of zein with lauryl chloride through an acylation reaction. The final products were confirmed by (1)H NMR, FT-IR analysis, and SDS-PAGE. Thermal analysis detected no microphase separation in the synthesized polymer matrix. As the content of lauryl moiety increased, the glass transition temperatures of modified zein decreased by as large as 25.8 °C due to the plasticization effect of the lauryl moiety. In addition, mechanical and surface properties of cast films from acylated zein were also investigated. The elongation at break of modified zein sheet was increased by about 7-fold at the high modification level with some loss of mechanical strength. The surfaces of modified zein films were as uniform as unmodified zein film but more hydrophobic, further suggesting that no microphase separation happened during the film formation process. This work indicated the potential of these new biomaterials in the development of biodegradable food packaging materials and delivery systems.     

Extraction and solubility characteristics of zein proteins from dry-milled corn

Zein isolation by aqueous ethanol extraction from dry-milled corn produces a mixture of zeins, covalently linked polymers (dimers, tetramers, etc.) and higher-molecular-weight aggregates, some of which were not soluble in aqueous alcohol. The insoluble particles were identified as protein aggregates which form when the extraction solution is heated, particularly under alkaline conditions. The insoluble protein aggregates were not present in zein isolated by the same method from corn gluten meal. Zeins extracted from corn gluten meal and dry-milled corn were fractionated (by differential solubility) to identify differences in their polypeptide compositions. Using polyacrylamide gel electrophoresis, beta- and gamma-zeins were detected in dry-milled corn, but only trace amounts of beta-zein were found in corn gluten meal. Treatment of dry-milled corn with 0.55% lactic acid and 0.2% sulfur dioxide at 50 degrees C for 6 h before ethanol extraction resulted in a 50% increase in zein isolate yield with high solubility (98%). This pre-extraction treatment cleaved disulfide linkages of the beta- and gamma-zeins and significantly reduced insoluble aggregates in zein isolates.     

Effects of Different Mill Types on Ethanol Production Using Uncooked Dry‐Grind Fermentation and Characteristics of Residual Starch in Distiller's Dried Grains (DDG)

This study aimed to investigate impacts of milling methods on ethanol production using an uncooked dry‐grind (cold fermentation) process and characterize residual starch in the distiller's dried grains (DDG) coproduct. Four corn lines with different chemical compositions were ground with cyclone, ultra‐centrifugal, or hammer mills equipped with a screen of 0.5 mm opening and used for the cold fermentation process. Greater starch hydrolysis and ethanol yield were obtained from cyclone‐milled corn, resulting from larger damaged starch contents and smaller particle sizes of the ground corn. Corn grains and ground corn after five‐month storage showed less starch hydrolysis than the freshly ground counterpart. Residual starch (2.8–8.0%) with large proportions of intact amylopectin contents (up to 42.5%) was found in the DDG from all types of milling. The results suggested that the entrapment of starch granules in ground corn and a low activity of amylolytic enzymes at a high ethanol concentration were accountable for the remaining of starch in the DDG.     

Water Vapor Barrier Properties of Zein Films Plasticized with Oleic Acid

Water sorption, water vapor permeability, and tensile properties were evaluated for zein films plasticized with oleic acid. The effect of relative humidity on water vapor permeability and tensile properties of films was investigated. Samples were produced by two different methods: casting from a zein solution and stretching from a zein-fatty acid resin. Films were also coated with linseed oil. Results indicated that preparation method affected water sorption and permeability of zein films. Resin films showed lower water sorption than cast films, especially at high A_w values. Water vapor permeability was also lower for resin films. Coating with linseed oil further improved water vapor barrier ability of resin films. Permeability was affected by environmental relative humidity; higher relative humidity resulted in increased permeability. Environmental relative humidity also affected tensile properties of resin films. Toughness and elongation were improved when relative humidity increased from 50 to 85% rh. Tensile strength showed a maximum at 75% rh. Coating improved elongation and toughness of films. Maximum elongation and toughness were observed for coated samples at 85% rh. Zein resin films showed good tensile and water barrier properties that were maintained through environmental humidity levels from 50 to 98% rh.     

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.     

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.     

Thermal Behavior of Zein Sheets Plasticized with Oleic Acid

Zein, extracted from underutilized corn gluten meal may serve as an alternative starting material for fabrication of biodegradable packaging. Zein plasticized with oleic acid may be formed into flexible and water‐resistant sheets. Our objective was to investigate the effect of plasticization on thermal behavior of zein sheets employing differential scanning calorimetry (DSC). Zein sheets were rolled from a resin prepared by dispersing zein and oleic acid in aqueous alcohol followed by the recovery of the formed moldable compound by precipitation with water. Sheets were later replasticized with additional oleic acid to increase ductility. DSC thermograms were used to investigate zein‐oleic acid interactions in zein sheets. DSC endotherms were attributed to melting of free oleic acid or to the dissociation of zein‐oleic acid bonds. Plasticized sheets contained bound and free oleic acid. Further heat‐treatment of plasticized sheets apparently resulted in zein absorption of free oleic acid. However, high temperatures were believed to cause dissociation of some zein‐oleic acid bonds formed during replasticization.