Effects of Certain Breadmaking Oxidants and Reducing Agents on Dough Rheological Properties'

Cereal Chem. 72(l):58-64 A dynamic rheometer was used to characterize the effect of glutathione, interchange. Addition of potassium bromate to dough resulted an increase potassium bromate, and two ascorbic acid isomers on the rheological in G'and a decrease in loss tangent. L-threoascorbic acid was rheologically properties of wheat flour doughs. During resting after mixing (dough more effective than D-erythroascorbic acid. This was explained by the relaxation), G' decreased and the loss tangent increased. The major factor presence of an active glutathione dehydrogenase in wheat flour that is causing those changes was suggested to be a sulfhydryl-disulfide interspecific for both glutathione and L-threoascorbic acid. change. Free radicals appeared to be involved in the sulfhydryl-disulfide Sperling (1986) cataloged the causes of stress relaxation into five categories: 1) a decrease in molecular weight caused by chain scission as a result of oxidative degradation or hydrolysis; 2) bond exchanges ongoing constantly in polymers, with or without stress (in the presence of a stress, however, the statistical rearrangements tend to reform the chains, so the stresses are reduced); 3) viscous flow caused by linear chains slipping past one another; 4) thirion relaxation as a reversible relaxation of the physical cross-links or trapped entanglements in elastomeric networks; 5) molecular relaxation, especially near the glass transition temperature (Tg), that tends to relieve any stress of chains during the experiment. The role of the sulfhydryl groups in dough chemistry has 'Contribution 94-432-J. Kansas Agricultural Experiment Station, Manhattan. 2 Graduate research assistant and professor, respectively, Department of Grain Science and Industry, Kansas State University, Manhattan. 3 Presently at Nabisco Brands, East Hanover, NJ. a 1995 American Association of Cereal Chemists, Inc. 58 CEREAL CHEMISTRY attracted the attention of many cereal chemists. The main premise has been that these sulfhydryl groups are potentially capable of undergoing a disulfide-sulfhydryl interchange that involves the cleavage or reformation of disulfide bonds mediated by sulfhydryl groups in flour or by relatively small amounts of added sulfhydryl compounds. Reduced glutathione (GSH) and oxidized glutathione (GSSG) are both naturally occurring in wheat flour (Kuninori and Matsumoto 1964, Hird et al 1968, Tkachuk 1969). Graveland et al (1978) reported that flour contained 5-7 mmol of sulfhydryl groups and 11-18 mmol of disulfide per kilogram. Kuninori and Sullivan (1968) studied disulfide-sulfhydryl interchange in wheat flour by adding radioactive glutathione. They reported that significant interchange took place in a flour-water dough, but not in a flour suspension. They postulated that mixing promoted the reaction of disulfide groups and GSH. Another possibility is that a free radical (GS-) is formed during mixing and may be involved in the disulfide-sulfhydryl interchange. Reaction with (GS-) can cause session of protein disulfide forming a protein thiyl radical. When interchain disulfide bonds are cleaved, the resulting depolymerization of the gluten proteins decreases the molecular weight and thereby reduces the elasticity and increases the extensibility of dough. Evidence for the occurrence of the interchain disulfide bonds in gluten, particularly in the glutenin fraction, have been presented by Beckwith and Wall (1966) and Yoshida et al (1980). Some of these interchain disulfide bonds may be important in maintaining the gluten structure: a reduction of 3-4% of the disulfide bond with mercaptoethanol caused a depolymerization of 80% of the high molecular weight gluten proteins (Jones et al 1974). It is generally accepted that the rheological properties of dough and its three-dimensional network are dependent on the arrangement and number of disulfide bonds and sulfhydryl groups of the protein. The vital contribution of disulfide bonds to dough stability has been shown in rheological studies by the addition of either sulfhydryl compounds or sulfhydryl-blocking reagents. A small amount of cysteine or reduced glutathione dramatically increases the extensibility of dough. Bloksma (1972) showed that both the viscous and elastic component of dough deformation were increased by reduced glutathione. Bloksma (1972) studied the relationship between the sulfhydryl and disulfide contents of dough and its rheological properties. He reported that only small fractions of the total sulfhydryl and disulfide groups were rheologically effective, and these fractions were much smaller than the chemically reactive ones. Jones et al (1974) estimated, on the basis of farinograph measurements, that -25-35% of sulfhydryl groups and 4-13% of the disulfide bonds were rheologically effective. However, a small decrease in crosslinking was sufficient to cause a considerable rheological effect because of disappearance of rheologically effective groups (Bloksma 1972). In most breadmaking processes, after mechanical development of the gluten network, the structure must be stabilized by oxidants. Minute amounts of oxidizing reagents such as potassium bromate or dehydroascorbic acid (DHAA) improve the handling and baking characteristics of wheat flour. Loaf volume increased, and bread had a better crumb grain (Jorgensen 1939). Most current theories on the improver action of oxidants agree that sulphydryl groups are involved in the reaction mechanism. The bromate is assumed to oxidize low molecular SHpeptides (glutathione) and consequently hamper sulfhydryldisulfide interchange of gluten molecules (Bloksma 1972). The sulfhydryl-disulfide interchange reaction also is used to explain the action of ascorbic acid, but the number of steps involved in its improver action are higher than with bromate. The four stereo isomers of ascorbic acid and their dehydro-forms display quite different activities as improvers. Walther and Grosch (1987) reported that the specificity of the enzyme glutathione dehydrogenase (dehydroascorbate reductase, EC 1.8.5.1) was responsible for the difference in the improver action. L-threodehydroascorbic acid was the best and D-threodehydroascorbic acid was the worst substrate of the four stereoisomers. This enzyme, which was discovered in wheat by Kuninori and Matsumoto (1963, 1964), oxidizes glutathione to its corresponding disulfide with DHAA as the oxidant. The enzyme appears to be specific for both glutathione and L-threodehydroascorbic acid. Thus, the improver action of L-threodehydroascorbic acid is explained by the oxidation of glutathione to the oxidized form. The decrease in glutathione decreases the rate of sulfhydryl-disulfide interchange in the dough. The order of substrate specificity of glutathione dehydrogenase for the four stereoisomers corresponds well with their improver action in dough (Mair and Grosch 1979). The purpose of this study was to characterize the effect of potassium bromate, glutathione, and ascorbic acid on the rheology of flour-water doughs using a dynamic rheometer. MATERIALS AND METHODS Commercial bread flour, 12.1% protein (N X 5.7), 0.45 ash, (14% mc) from Ross Industries (Cargill, Wichita, KS) was used. The flour was fractionated into water-insoluble and soluble fractions according to the procedure shown in Figure 1. One part of flour was suspended in three parts of distilled water and stirred continuously for 15 min. Then the suspension was centrifuged for 20 min at 1,000 X g. The insoluble residue, gluten plus starch, was frozen and lyophilized. The water-soluble fraction was boiled for 15 min and then frozen and lyophilized. Both the water-insoluble and water-soluble fractions were ground in