Extraction and Film Properties of Kafirin from Coarse Sorghum and Sorghum DDGS by Percolation

The high cost of kafirin and zein restricts their use for bioplastic and food applications. Effective, simple, and rapid kafirin/zein isolation processes are required. Here a percolation‐type aqueous ethanol solvent extraction process from coarse meals (grits) and coarse sorghum distillers dried grains and solubles (DDGS) for kafirin and zein isolation employing a low ratio of extractant to meal (2.5:1) was investigated, which is potentially applicable in the grain bioethanol industry. Postextraction filtration times were more than twice as fast using coarse meals compared with fine flours. Washing the meals prior to extraction to remove starch improved protein preparation purity to 73–85% compared with 68–72% for unwashed meals. Hence, no subsequent filtration or centrifugation step is required to clean up the kafirin/zein solution prior to solvent evaporation. With a single extraction step, kafirin/zein yields were 48% (protein basis) for DDGS and 53–70% for washed sorghum/maize meals. Cast films were used as a model bioplastic system to evaluate extracted kafirin/zein functional properties. DDGS kafirin films had rough surfaces but had the lowest water uptake and in vitro digestibility, owing to heat‐induced disulfide crosslinking during DDGS processing. Extraction by percolation using coarse meal/DDGS has potential to improve kafirin/zein viability.     

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.     

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.     

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of zeins in mature maize kernels

A high-throughput method has been developed to allow rapid analysis of maize seed storage proteins by matrix-assisted laser desorption time-of-flight mass spectrometry. The extraction solution containing an organic solvent, a reducing agent, and a volatile base has been optimized to enable extraction of all classes of zein proteins (alpha-, beta-, gamma-, and delta-). A near-saturating concentration of matrix, 2-(4-hydroxyphenylazo)benzoic acid, was necessary to obtain strong peaks for the most lipophilic zeins, the alpha-zeins. Zein proteins with small mass differences, difficult to separate by sodium dodecyl sulfate polyacrylamide gel electrophoresis, were resolved through this analysis. Mass signals corresponding to the 10-kDa delta-, 15-kDa beta-, 16-kDa gamma-, 27-kDa gamma-, and several 19 and 22-kDa alpha-zeins were detected. The zein identities were further confirmed by the association of the number of cysteine residues in each zein MS peak, as determined by iodoacetamide derivatization, with the number predicted from its coding sequence. The relative zein abundance in the zein MS peaks was also correlated with the relative zein EST abundance among endosperm EST libraries. This method was utilized to examine the zein composition of a number of corn inbred lines and opaque mutants.     

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.     

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%).     

Selected Factors Affecting Crude Oil Analysis of Distillers Dried Grains with Solubles (DDGS) as Compared with Milled Corn

With increasing production of distillers dried grains with solubles (DDGS), both fuel ethanol and animal feed industries are demanding standardized protocols for characterizing quality. AOCS Approved Procedure (Am 5‐04) was used for measuring crude oil content in milled corn and resulting DDGS. Selected factors, including sample type (milled corn, DDGS), sample origin (ethanol plant 1, 2, 3), sample particle size (original matrix, <0.71 mm, <0.50 mm mesh opening; the last two materials were obtained by grinding and sieving), solvent type (petroleum ether, hexane), extraction time (30, 60 min), and postextraction drying time (30, 60 min) were investigated by a complete factorial design. For milled corn, only sample origin and extraction time had significant effects (P < 0.05) on crude oil values measured, but for DDGS, besides those two factors, sample particle size, solvent type, and drying time also had significant effects. Among them, the particle size of DDGS had the most effect. On average, measured oil content in DDGS ranged from 11.11% (original matrix) to 12.12% (<0.71 mm) and to 12.55% (<0.50 mm). For measuring the crude oil content of DDGS, particle size reduction, 60 min of extraction, and 60 min of drying are recommended. Regardless of the underlining factors, the method was very repeatable (standard errors <0.05). The observed particle size effect on crude oil analysis of DDGS suggests the need for similar confirmations using other analytical methods.     

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.     

Effect of Fractionation of Distillers Dried Grains With Solubles (DDGS) on Pelleting Characteristics of Broiler Diets

Recently, the Elusieve process, a combination of elutriation (air classification) and sieving (screening) was developed to separate fiber from distillers dried grains with solubles (DDGS) to increase DDGS utilization in nonruminant (poultry and swine) diets. Elusieve process produces three products: 1) Pan DDGS, with 5% higher protein content than conventional DDGS, which would be used at higher inclusion levels in broiler diets because of low fiber content; 2) Big DDGS, with nearly the same protein content as conventional DDGS, which would be used at same inclusion levels as conventional DDGS; and 3) Fiber product. The objective of this study was to determine and compare pellet‐mill throughput, power consumption, and pellet quality for broiler diets incorporating different levels (0, 10, and 20%) of conventional DDGS and DDGS products from Elusieve process. Poultry oil contents were lower (1.5–1.6%) in diets comprising Pan DDGS and diets without DDGS than in the other diets (2.2–3.1%). The feed throughput was not affected by inclusion levels or type of DDGS. Pellet quality (pellet durability index [PDI]) for diets comprising Pan DDGS (both 10 and 20% inclusion levels) was significantly better than PDI for diets comprising conventional DDGS, Big DDGS, and the diet without DDGS. Better pellet quality of diets comprising Pan DDGS could be due to lower quantity of poultry oil used as well as compositional characteristics such as low fiber and high protein. Diets with Big DDGS had similar pelleting characteristics to those with conventional DDGS. Pellet quality deteriorated at higher inclusion levels of conventional DDGS, Big DDGS, and Enhanced DDGS. Considering that Pan DDGS would be included at higher inclusion levels in broiler diets, superior pellet quality of diets comprising Pan DDGS is beneficial.     

Economics of Fiber Separation from Distillers Dried Grains with Solubles (DDGS) Using Sieving and Elutriation

Separation of fiber from distillers dried grains with solubles (DDGS) provides two valuable coproducts: 1) enhanced DDGS with reduced fiber, increased fat and increased protein contents and 2) fiber. Recently, the elusieve process, a combination of sieving and elutriation was found to be effective in separating fiber from two commercial samples of DDGS (DDGS‐1 and DDGS‐2). Separation of fiber decreased the quantity of DDGS, but increased the value of DDGS by increasing protein content and produced a new coproduct with higher fiber content. Economic analysis was conducted to determine the payback period, net present value (NPV), and internal rate of return (IRR) of the elusieve process. The dependence of animal foodstuff prices on their protein content was determined. Equipment prices were obtained from industrial manufacturers. Relative to crude protein content of original DDGS, crude protein content of enhanced DDGS was higher by 8.0% for DDGS‐1 and by 6.3% for DDGS‐2. For a dry‐grind plant processing corn at the rate of 2,030 metric tonnes/day (80,000 bushels/day), increase in revenue due to the elusieve process would be $0.4 to 0.7M/year. Total capital investment for the elusieve process would be $1.4M and operating cost would be $0.1M/year. Payback period was estimated to be 2.5–4.6 years, NPV was $1.2–3.4M, and IRR was 20.5–39.5%.     

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-, β-, 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). β-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.     

Development of New Method for Extraction of α‐Zein from Corn Gluten Meal Using Different Solvents

A modified procedure for the extraction of α‐zein from corn gluten meal was developed and compared against a commercial extraction method. The modification involved raising the concentration of alcohol in solvent and removing the precipitate by centrifugation. Five organic solvent mixtures were compared using the modified extraction procedure developed along with the reductant sodium bisulfite and NaOH. The modified procedure precipitated most of the non‐α‐zein protein solids by increasing the concentration of alcohol. The supernatant had α‐zein‐rich fraction, resulting in higher yield of α‐zein than the commercial method when cold precipitated. The commercial extraction procedure had a zein yield of 23% and protein purity of 28% using 88% 2‐propanol solvent. The three best solvents, 70% 2‐propanol, 55% 2‐propanol, and 70% ethanol, yielded ≈35% of zein at protein purity of 44% using the modified extraction procedure. Zeins extracted using the novel method were lighter in color than the commercial method. Densitometry scans of SDS‐PAGE of α‐zein‐rich solids showed relatively large quantities of α‐zein with apparent molecular weights of 19,000 and 22,000 Da. The α‐zein‐rich solids also had small amounts of δ‐zein (10,000 Da) because it shares similar solubility properties to α‐zein. A solvent mixture with 70% 2‐propanol, 22.5% glycerol, and 7.5% water extracted significantly less zein (≈33%) compared to all other solvents and had α‐zein bands that differed in appearance and contained little to no δ‐zein.     

Effect of DDGS,Moisture Content,and Screw Speed on Physical Properties of Extrudates in Single‐Screw Extrusion

Three isocaloric (3.5 kcal/g) ingredient blends containing 20, 30, and 40% (wb) distillers dried grains with solubles (DDGS) along with soy flour, corn flour, fish meal, and mineral and vitamin mix, with net protein adjusted to 28% (wb) for all blends, were extruded in a single‐screw laboratory‐scale extruder at screw speeds of 100, 130, and 160 rpm, and 15, 20, and 25% (wb) moisture content. Increasing DDGS content from 20 to 40% resulted in a 37.1, 3.1, and 8.4% decrease in extrudate durability, specific gravity, and porosity, respectively, but a 7.5% increase in bulk density. Increasing screw speed from 100 to 160 rpm resulted in a 20.3 and 8.8% increase in durability and porosity, respectively, but a 12.9% decrease in bulk density. On the other hand, increasing the moisture content from 15 to 25% (wb) resulted in a 28.2% increase in durability, but an 8.3 and 8.5% decrease in specific gravity and porosity, respectively. Furthermore, increasing the screw speed and moisture content of the blends, respectively, resulted in an increase of 29.9 and 16.6% in extruder throughput. The extrudates containing 40% DDGS had 8.7% lower brightness, as well as 20.9 and 16.9% higher redness and yellowness, compared with the extrudates containing only 20% DDGS. Increasing the DDGS content from 20 to 40% resulted in a 52.9 and 51.4% increase in fiber and fat content, respectively, and a 7.2% decrease in nitrogen free extract. As demonstrated in this study, ingredient moisture content and screw speed are critical considerations when producing extrudates with ingredient blends containing DDGS, as they are with any other ingredients.     

Total Phenolics and Antioxidant Activity of Water and Ethanolic Extracts from Distillers Dried Grains with Solubles With or Without Microwave Irradiation

The purpose of this study was to determine the efficacy of extracting phenolic compounds with antioxidant activity from distillers' dried grains with solubles (DDGS) with water, 50% aqueous ethanol, and absolute ethanol, using microwave irradiation or a water bath at various temperatures. DDGS was extracted for 15 min with each solvent while heating at 23, 50, 100, and 150°C by microwave irradiation or in a water bath at 23, 50, and 100°C. Phenolic content of extracts increased with increasing temperature to a maximum of 12.02 mg/g in DDGS extracts that were microwave irradiated in water or with 50% aqueous ethanol at 150°C. Antioxidant activity range was 1.49–6.53 μmol of Trolox equivalents/g of DDGS. Highest antioxidant activities were obtained from 50% aqueous ethanol extracts at all temperatures, and water extracts that were heated at 100 and 150°C. These data indicate that DDGS extracts with high phenolic content and antioxidant activity can be obtained from DDGS, particularly with the use of water or 50% ethanol and high temperature (100 or 150°C). This may be valuable to ethanol manufacturers, livestock producers, and food and nutraceutical companies.     

Methods for Characterization of Residual Starch in Distiller's Dried Grains with Solubles (DDGS)

The objective of this study was to establish methods for determining the content and components of residual starch in distiller's dried grains with solubles (DDGS), a coproduct from dry‐grind corn ethanol production. Four DDGS prepared in our laboratory and one DDGS obtained from a commercial ethanol manufacturer were used for the study. Quantitative analysis of total residual sugar (TRS) in DDGS was performed by determining d ‐glucose produced by enzymatic hydrolysis of oligosaccharides and residual starch remaining in hexane‐defatted DDGS after being dispersed in 90% DMSO. The TRS consisted of free glucose, oligosaccharides, and residual starch. The commercial manufacturer's DDGS contained more TRS (15.8%, w/w db) than the laboratory‐processed DDGS (2.4–2.9%, w/w db). The content of residual starch remaining in the commercial DDGS (5.5% w/w db) was also larger than the laboratory‐processed DDGS (1.9–2.5% w/w db). Analyses of molecular weight distribution showed that the residual starch in DDGS consisted of short‐chain amylose and amylopectin, respectively, as the major and minor components. The short‐chain amylose molecules constituted 86.5–94.1% of the residual starch. The major population of the short‐chain amyloses had an average degree of polymerization (DP) of 85, closely resembling the length of enzyme‐resistant fragments of amylose‐lipid complexes.     

Separation of Fiber from Distillers Dried Grains with Solubles (DDGS) Using Sieving and Elutriation

A process was developed to separate fiber from distillers dried grains with solubles (DDGS) in a dry‐grind corn process. Separation of fiber from DDGS would provide two valuable coproducts: 1) DDGS with reduced fiber, increased fat, and increased protein contents; and 2) fiber. The process, called elusieve process, used two separation methods, sieving and elutriation, to separate the fiber. Material carried by air to the top of the elutriation column was called the lighter fraction and material that settled to the bottom of the column was called the heavier fraction. We evaluated the compositions of fractions produced from sieving and elutriation. Two commercial samples of DDGS were obtained from two dry‐grind corn plants. Sieving over four screens (869, 582, 447, and 234 μm openings) created five size categories. The two smallest size categories contained >40% (w/w) of the original DDGS and had reduced fiber and increased protein and fat contents relative to the original DDGS. Elutriation of the remaining three size categories increased protein and fat contents and reduced fiber contents in the heavier fractions. Elutriation at air velocities of 1.59–5.24 m/sec increased the protein content of the heavier fraction by 13–41% and increased the fat content of the heavier fraction by 4–127% compared with the bulk fractions of each size category. This process was effective in separating fiber from both DDGS samples evaluated. Elusieve process does not require changes in the existing dry‐grind process and can be implemented at the end of the dry‐grind process.     

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).     

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.     

Use of Phytases in Ethanol Production from E‐Mill Corn Processing

Effects of phytase addition, germ, and pericarp fiber recovery were evaluated for the E‐Mill dry grind corn process. In the E‐Mill process, corn was soaked in water followed by incubation with starch hydrolyzing enzymes. For each phytase treatment, an additional phytase incubation step was performed before incubation with starch hydrolyzing enzymes. Germ and pericarp fiber were recovered after incubation with starch hydrolyzing enzymes. Preliminary studies on phytase addition resulted in germ with higher oil (40.9%), protein (20.0%), and lower residual starch (12.2%) contents compared to oil (39.1%), protein (19.2%), and starch (18.1%) in germ from the E‐Mill process without phytase addition. Phytase treatment resulted in lower residual starch contents in pericarp fiber (19.9%) compared to pericarp fiber without phytase addition (27.4%). Results obtained led to further investigation of effects of phytase on final ethanol concentrations, germ, pericarp fiber, and DDGS recovery. Final ethanol concentrations were higher in E‐Mill processing with phytase addition (17.4% v/v) than without addition of phytase (16.6% v/v). Incubation with phytases resulted in germ with 4.3% higher oil and 2.5% lower residual starch content compared to control process. Phytase treatment also resulted in lower residual starch and higher protein contents (6.58 and 36.5%, respectively) in DDGS compared to DDGS without phytase incubations (8.14 and 34.2%, respectively). Phytase incubation in E‐Mill processing may assist in increasing coproduct values as well as lead to increased ethanol concentrations.