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

Cereal Chem. 88(4):356-362 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% 2propanol, 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. Zein is ≤52% protein by weight in the corn kernel. Zein proteins were first described by Gorham (1821), who extracted them from Indian corn. Interest in the protein developed when Osborne (1891) extracted zein from corn gluten meal (CGM), which was a high-protein coproduct of corn wet milling. Later, Osborne and Mendel (1914) classified protein from corn into four different categories based on solubility. These proteins were albumins (soluble in pure water), globulins (soluble in aqueous salt solutions), prolamins (soluble in 70% ethanol), and glutelins (soluble in dilute acid or base). Zein proteins are prolamins and have been further characterized into four different classes based on solubility, electrophoresis, and immunological studies. Esen (1987, 1990) classified them as α, β, γ, and δ-zein. Important past applications of zein were in inks, adhesives, coatings, plastics, and chewing gums (Sturken 1938; Coleman 1939, 1941; Lougovoy 1949; Simonds et al 1949). New potential applications of zein include packaging, carrier material for chemical or drug delivery, and biomedicine such as cellular scaffolds to accelerate cell growth in tissue and bone, while degrading after healing (Dong et al 2004; Wang et al 2007; Tu et al 2009; Jiang et al 2010). It is important to recover higher amounts of functional zein fractions with higher purity to suit potential novel applications. The first commercial extraction of zein was made in 1939 (Shukla and Cheryan 2001). A refined patent detailed the commercial extraction process for zein using 85% aqueous 2-propanol at 60°C (Swallen 1942). The extract was then treated with hexane to remove pigment; the zein was collected by precipitation in cold water and drying on ring dryers. A current commercial method by Carter and Reck (1970) extracts zein from CGM with 88% (w/w) 2-propanol at 65°C with agitation. The extract is cold-precipitated at –10 to –20°C to precipitate α-zein and dried in a vacuum oven. To produce higher purity zein protein, the cold-precipitated wet solids could be redissolved in the extracting solvent and reprecipitated. The zein extraction method of Carter and Reck (1970) also used 0.25% NaOH to adjust the CGM extraction from pH 6.5 to 7.0, which is near the isoelectric point of many zein phenotypes, causing them to become insoluble while leaving α-zein soluble (Cook et al 1996; Carter and Reck 1970). Another commercial extraction procedure was described by Takahashi and Yanai (1994), who extracted α-zein from CGM using 70% (v/v) aqueous acetone at 40°C. The extract was then concentrated and added to absolute acetone to precipitate the α-zein protein. These current commercial extraction methods extract primarily α-zein, which is soluble in aqueous alcohol solvents at higher alcohol concentrations such as 88% (w/w) 2-propanol (Esen 1987; Kale et al 2007). A major problem with these methods is low zein yields of 22 and 20.4 g/g of CGM (db) reported by Carter and Reck (1970) and Takahashi and Yanai (1994), respectively. These yields are low considering that >50% of the α-zein is not extracted (Wu et al 1997b). Zein extraction yields could be improved by using lower aqueous alcohol concentrations such as 55% (w/w) 2-propanol. This increase of yield would be due to coextraction of all zein fractions resulting in lower α-zein purity (Esen 1986). However, lower alcohol concentrations decrease zein solubility and solution stability (Swallen 1942). Other issues with both types of solvents and extraction systems are 1) the large amounts of solvents required, and 2) the energy intensive processes such as solvent concentration, cold-precipitation, and distillation of solvent for recycling. The purpose of this research was to modify the Carter and Reck (1970) zein extraction procedure to improve the yield and purity of the zein protein using reductant and different solvents. The solvents in this experiment were selected based on known information about the structure of zein protein bodies and solubilities. Zein protein bodies are ≈1 μm, can survive grinding and mild cooking, and subsequently are intact in corn flour (BattermanAzcona and Hamaker 1998). The making of CGM consists of grinding and low temperature processing that disperses the endosperm starch and protein matrix to produce intact starch and zein protein bodies (Cox et al 1944; Wu et al 1997a). The zein protein bodies consist of a thin layer of βand γ-zein interconnected by disulfide bonds. This layer sheaths a large proportion of α-zein at the protein body’s core (Mohammad and Esen 1990). One of the commercial extraction methods uses 88% (w/w) aqueous 2-propanol, 0.25% NaOH, and no reducing agent (Carter and Reck 1970). The 88% (w/w) aqueous 2-propanol dissolves α-zein across the layer without dissolving βand γ-zeins (Carter and Reck 1970). For this research, solvents that extract total zein, such as 55% (w/w) aqueous 2-propanol and 70% (v/v) aqueous ethanol, were chosen. 1 Graduate student, Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011. 2 Assistant professor, Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011. 3 Corresponding author. Phone: (515) 294-8681. Fax: (515) 294-8181. E-mail: