756 CEREAL CHEMISTRY Extrusion Chemistry of Wheat Flour Proteins: II. Sulfhydryl-Disulfide Content and Protein Structural Changes C. A. Rebello 1,2 and K. M. Schaich 1,3 ABSTRACT Cereal Chem. 76(5):756–763 Effects of twin-screw extrusion conditions on wheat flour proteins were studied, using a two-level fractional factorial experimental design (11 and 14% protein content, 160 and 185°C, 16 and 20% moisture, 300 and 500 rpm screw speed, mass flow rate of 225 and 400 g/min). Total pro- tein detectable by solid-phase bicinchoninic acid assay decreased slightly after extrusion, with greatest protein loss at 16% moisture and 160°C. Sulfhydryl content of both flours increased after extrusion at 185°C and 16% moisture with moderate specific mechanical energy (SME ≈ 400–600 kJ/kg) or 160°C and 16% moisture with high SME (SME > 1,000 kJ/kg). Disulfide bonds increased under comparable conditions but with moderate shear (SME = 510–540 kJ/kg). At 20% moisture and either temperature, sulfhydryl and total thiol contents decreased without corres- ponding increases in disulfides. Reversed-phase HPLC indicated gliadins were the fractions most affected by extrusion; high molecular weight glutenin subunits also were affected. Changes in gliadins were extensive at 185°C and 16% moisture and were minimal at 160°C and 20% mois- ture. SDS-PAGE confirmed the disappearance of protein bands and ap- pearance of new material at low and high molecular weights, presumably resulting from polypeptide fragmentation followed by random radical re- combination. Both protein fragmentation and cross-linking appeared to in- volve free radicals. Extrusion technology plays a central role in modern cereal-based industry, and significant advances in engineering have led to pro- duction of a wide range of extruded products. Nevertheless, sur- prisingly little still is known about the basic molecular phenomena that contribute to texturization and other product characteristics during extrusion of wheat flour products. Early studies on extrusion of wheat flour suggest that the mechan- ical and textural properties of extrudates are largely determined by gluten proteins in dough (Hauck 1980). The mechanical strength of extrudates is directly related to the gluten content of flour (Linko et al 1981). Faubion and Hoseney (1982a,b) extruded wheat starch with varying amounts (1–16%) of added wheat gluten or soy protein isolate and showed that the presence of protein is crit- ically important for texturization and that the amount and type of protein in wheat flour markedly affects the textural properties of extrudates. Expansion decreased steadily as gluten protein content increased from 1 to 11%; at levels higher than 11%, expansion again increased. Shear and break strengths changed correspondingly. Sim- ilar effects were produced when yeast protein concentrate was ex- truded with wheat starch (Lai et al 1985). However, addition of soy proteins had the opposite effect: increasing expansion with protein levels of up to 8% and decreasing expansion with protein levels higher than 10%. Extrudates containing soy protein showed sub- stantially greater expansion than those containing wheat gluten. These results indicate that both the type and level of protein exert important effects on the physical properties of extruded cereal products. However, relatively little is known about the specific mo- lecular changes responsible for these properties. Studies on soy flours and concentrates have attributed texturization to cross-linking of soy proteins, involving both main-chain polypeptide and disulfide bonds (Cumming et al 1973, Hansen et al 1975, Burgess and Stanley 1976, Hager 1984, Neumann et al 1984), although the relative con- tributions of the two types of cross-linking have not been distin- guished. In contrast, information about molecular changes in wheat proteins during extrusion is limited. Electron paramagnetic resonance (EPR) studies have demonstrated the presence of nitrogen- and sulfur-centered radicals during extru- sion of wheat flour (Koh et al 1996, Schaich and Rebello 1999). The presence of these radicals suggests fragmentation at disulfide bonds and main-chain peptide or side-chain amide bonds, and radical recom- binations could be responsible for cross-linking in extruded products. The chemistry potentially associated with free radical production in proteins of extruded wheat flour should be traceable. We report the effects of extrusion on protein thiol-disulfide content and changes in protein molecular weight arising from fragmentation and cross-linking. Correlation of these chemical changes with free radical production in wheat flour extrudates is demonstrated. MATERIALS AND METHODS Two commercial wheat flours were obtained from Bay State Mil- ling Co. (Minneapolis, MN): Bouncer (14% protein, 0.52% ash, and 14% moisture) and Boss (11.4% protein, 0.41% ash, and 14% mois- ture). Both flours were mixtures of hard red wheat cultivars. Bouncer was composed of spring wheats, and Boss was composed of win- ter wheats containing up to 50% Bouncer. The flours were chosen for evaluation and comparison of extrusion behavior because they are commonly used in breadmaking. Protein levels were selected to bracket (low and high) the useful functional range shown by Fau- bion and Hoseney (1982b) for such flours. Bicinchoninic acid (BCA) test reagents for determination of protein were purchased from Pierce Chemical Co. (Rockford, IL). 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) for free sulfhydryl and total thiol assays were obtained from Aldrich Chemical Co. (Milwaukee, WI). 4-Vinyl pyridine, trichloroacetic acid, and 2- mercaptoethanol (Sigma Chemical Co., St. Louis, MO) were used in reversed-phase (RP) HPLC of wheat proteins. SDS (BioRad Laboratories, Richmond, CA); ISS-Pro-Blue staining solution, dithioerythritol, Daiichi silver stain (Integrated Separation Systems, Natick, MA); and acrylamide and bisacrylamide (Amresco Corp., Solon, OH) were used in PAGE of wheat flour proteins. All reagents used in extractions and analyses were of the highest grade available. Extrusion Conditions Extrusion of the two wheat flours was conducted on a twin- screw extruder (ZSK30, Werner and Pfleiderer, Ramsey, NJ) as de- scribed in Schaich and Rebello (1999). Chemical Analyses Extrudates were ground in a mill (Micro Wiley, Thomas Scien- tific, Swedesboro, NJ) to pass through a 40-mesh sieve. Ground extrudates were placed in glass containers, flushed with nitrogen, 1 Department of Food Science, Rutgers University, 65 Dudley Rd., New Brunswick, NJ 08901-8520. 2 Current address: Campbell Soup Company, Campbell Place, Camden, NJ 08103. 3 Corresponding author. Phone: 732/932-9611, ext. 233; Fax: 609/497-9313; E-mail: schaich@aesop.rutgers.edu. Publication no. C-1999-0804-05R. © 1999 American Association of Cereal Chemists, Inc.