Wheat Proteins Extracted from Flour and Batter with Aqueous Ethanol at Subambient Temperatures George H. Robertson," 2 Trung K. Cao,' and William J. Orts' ABSTRACT Cereal Chem. 84(5):497-501 Contact of wheat flour with aqueous ethanol may enrich protein by starch displacement or deplete protein by extraction depending on I) extraction conditions and 2) the form of the substrate. Extraction at sub- ambient temperatures has not been described for specific gliadins for either dry flour with the protein in native configurations or for wet, developed hatter or dough. This limits the ability to interpret technologies such as the cold-ethanol method. Here, we describe specific albumin and gliadin composition of flour extracts by capillary zone electrophoresis CZE in 0-100% (v/v) ethanol from -12 to 22°C. Extraction was reduced for albumin and gliadin protein as the temperature was reduced and the concentration range for extraction narrowed. Extraction dropped precipi- tously between 0 and -7°C for both albumins and gliadins. Electrophor- etically defined gliadins extracted in constant proportion at 22°C and 30- 80%(v/v) ethanol, but at lower temperature, the u-gliadins were enriched and 13-gliadins depleted in the 30-55% (v/v) range. For extracts from wheat flour batter, depletion of a and 13 and enrichment of y relative to the dry flour contact suggested that the electrophoretically slow migrating y- and (1)-proteins are less well incorporated to the dough matrix than electrophoretically fast migrating (x and 13 types. Aqueous ethanol solutions have been exploited for large-scale refining of substrates as diverse as human blood, wood pulp, and oilseeds (Cohn et al 1946; Oncley et al 1949; Rao et al 1955; Rao and Arnold 1956a,b, 1957, 1958; Ni and Van Heiningen 1996). Substrates such as cereal grains, cottonseed, plywood, and plant straw also may be refined using ethanol, but this has been limited to laboratory or pilot scale (Hron and Koltun 1984; Hojilla-Evan- gelista et al 1992a,b; Chang et al 1995; Papatheofanous et al 1995; Robertson and Cao 1998a,b; Feng et al 2002; Miller et al 2002) The attractiveness and utility of aqueous ethanol as a proces- sing/separation agent arises from its unique physical and thermo- dynamic properties. its low heat capacity, latent heats of crystal- lization and evaporation, boiling temperature, and density translate to reduced energy usage, fewer undesirable heat-induced property changes, and improved settling rates for crop components (Robert- son and Otis 2005). However, management of the concentration, temperature, pH, or ionic strength enhances or inhibits the ability to extract water, oil, lignin, or proteins. Management of these factors implies the technical ability to fully or partially remove and recover soluble and suspended substances so that the ethanol may be recycled. Adding to the interest in ethanol facilitated re- fining is the emergence of grain-based, ethanol-producing biorefin- eries. In these facilities, there is also a need for improved, energy- efficient component fractionation to recover high-value lipids and proteins to bolster the economics of the enterprise. However, the use of aqueous ethanol to refine diverse biological materials creates the need to understand the selectivity for components in the biological substrate. This knowledge can help to identify poten- tial refining options and product-platform opportunities. Research in our laboratory helps to define some issues related to ethanol use in refining. We have applied refrigerated or cold ethanol to the refining of wheat grain to produce starch and protein fractions as "platforms" for well-developed food markets as well as for emerging food and nonfood markets. In this practice, cold ethanol was applied to a mechanically developed batter while the batter was physically manipulated. The liquid ethanol solution separates starch and protein by extracting water and physically displacing starch from the protein matrix. We found that if the solution and the batter were refrigerated to -15 and 10°C, respec- United States Department of Agriculture, Pacific West Area, Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710. 2 Corresponding author. E-mail: grobertson@pw.usda.gov doi: 10.1 094/CCHEM-84-5-0497 This article is in the public domain and not copyrightable. It may be freely re- printed with customary crediting of the source. AACC International, Inc., 2007. lively, excellent separation and recovery were possible because the solubility of the gluten protein is low, as confirmed by total protein analysis. However, severe and extended mechanical action during the displacement step may cause selective loss of gliadin- class protein to solution. Furthermore, we found functional differ- ences in the protein gluten fraction produced by the cold ethanol method (Robertson and Cao 2001, 2002, 2003, 2004). It is unknown whether the differences in extracted proteins orgi- nate in the native molecular structures or in the hydrated structures created to affect the starch protein separation. For wheat gliadins, in particular. we note only the plethostatic or cloud-point determi- nation of critical solubility of previously fractionated Osborne gliadins (Osborne 1907; Dill and Alsberg 1925). The plethostatic method defines the critical temperature at which turbidity initiates on cooling of previously heated and clear protein solutions. The method is best applied to and interpreted for single components in solution. However, wheat proteins do not fit this purity constraint so that the method may index the precipitation of only the least soluble gliadin subcomponent. Above the critical solubility temper- ature, all gliadin-class proteins were described as fully soluble. We also note reports of solubility of gliadins and whole gluten at 70% (v/v) ethanol and 20°C extraction (Meredith et al 1960a,b; Meredith 1965a,b,c; Robertson et al 1999) and gluten protein solu- bility at 22°C in ethanol solutions from 0 to 100% (vlv) (Robertson and Cao 2001, 2002, 2003, 2004). In the present study, we describe the dissolved flour proteins in extracts of native molecular matrices at ambient and subambient temperatures in water, ethanol, and ethanol-water solutions. A number of sophisticated methods have been developed for analysis of proteins in wheat, including reversed-phase HPLC, capillary electrophoresis, and a variety of gel-electrophoresis techniques (Shewiy and Lookhart 2003). Capillary zone electrophoresis (CZE) separates proteins by a voltage-driven migration in acidic buffer based on charge density. Highly charged, small diameter proteins have the greatest mobility in the analysis and are detected first. This automated electrophoretic method has been used successfully for the direct analysis of wheat gliadins and albumins in Osborne- like ethanol extracts employing a polymer-modified acidic buffer. Furthermore, capillary electrophoresis is known to be comple- mentary to reversed-phase HPLC, which separates on the basis of surface hydrophobicity. Some single HPLC peaks have been re- solved into multiple CZE peaks. The method requires miniscule amounts of buffer and no solvent, thereby maximizing safety, min- imizing environmental hazard, and reducing material and waste disposal costs (Lookhart and Bean 1995a,b; Robertson and Cao 2004). Only filtration and centrifugation were needed to prepare Vol. 84, No. 5, 2007 497