1 This is the final draft post-refereeing. 2 The publisher’s version can be found at http://dx.doi.org/10.1016/j.jcs.2006.03.003 3 4 5 Please cite this article as: Lagrain, B., Brijs, K., Delcour J.A. Impact of redox agents on the physico-chemistry of wheat gluten proteins during hydrothermal treatment, Journal of Cereal Science 2006, 44, 49-53. 6 7 Impact of redox agents on the physico-chemistry of wheat gluten proteins during 8 hydrothermal treatment 9 10 Bert Lagrain*, Kristof Brijs and Jan A. Delcour 11 Laboratory of Food Chemistry, Katholieke Universiteit Leuven 12 Kasteelpark Arenberg 20, B-3001 Leuven, Belgium 13 14 *Corresponding author: 15 Bert Lagrain 16 Tel: + 32 (0) 16321634 17 Fax: + 32 (0) 16321997 18 E-mail address: bert.lagrain@biw.kuleuven.be 19 20 1 21 Abbreviations: db, dry basis; DTT, dithiothreitol; HPLC, high performance liquid 22 chromatography; HT, holding time; P, Poise; RVA, rapid visco analyser; SDS, sodium 23 dodecyl sulphate; SE, size exclusion; SH, sulphydryl. 24 25 26 Keywords: RVA, Wheat gluten, Heat treatment, Protein extractability, Oxidants, 27 Reducing agents 28 2 29 Abstract 30 The impact of the oxidants potassium bromate and potassium iodate and the reducing 31 agent dithiothreitol (DTT) on the rheological behaviour of 20% (w/v) gluten-in-water 32 suspensions during thermal treatment was monitored with the rapid visco analyser 33 (RVA). The suspensions were subjected to a linear temperature increase from 40 °C to 34 95 °C in 14 min, a holding step of 40 min at 95 °C, a cooling step (7 min) with a linear 35 temperature decrease to 50 °C, and a final holding step at 50 °C (13 min). Potassium 36 iodate (1.18 and 1.77 µmol/g protein) and potassium bromate (1.52 and 15.2 µmol/g 37 protein) decreased RVA viscosities in the holding step and increased sodium dodecyl 38 sulphate (SDS) protein extractabilities suggesting a greater heat resistance and decreased 39 gliadin-glutenin cross-linking. In contrast, DTT (1.65 and 3.30 µmol/g protein) made 40 RVA viscosity increase at lower temperatures and lowered SDS extractabilities. It is 41 postulated that low concentrations of reducing agent facilitate gliadin-glutenin cross- 42 linking during heating while oxidants hinder gluten polymerization due to decreased 43 levels of free sulphydryl groups and less flexibility of the glutenin chains. 44 3 45 1. Introduction 46 The storage proteins of wheat (consisting of monomeric gliadins and polymeric 47 glutenins) are of great importance in bread making. During dough mixing, these flour 48 proteins form a visco-elastic gluten network which is responsible for dough gas retention 49 during proofing and contributes to the final bread structure. Gluten proteins polymerize 50 during heat treatment. Sulphydryl (SH)-disulphide interchange reactions induced by heat 51 affect all gluten proteins except the cysteine free ω-gliadins (Booth et al., 1980; Schofield 52 et al., 1983) and result in the incorporation of gliadin monomers in the glutenin network 53 (Morel et al., 2002; Redl et al., 1999; Singh and McRitchie, 2004). Lagrain et al. (2005) 54 applied a temperature profile and simultaneously measured rheological changes in a 55 gluten-water suspension using the Rapid Visco Analyser (RVA). Both gliadin and 56 glutenin contributed to the initial viscosity in the RVA profile. When the temperature was 57 increased to 95 °C, glutenin extractability decreased. Holding at 95 °C resulted in 58 polymerization of both gliadin and glutenin. Above 80 °C, the RVA viscosity steadily 59 increased with longer holding times while the extractability of gliadin and glutenin 60 decreased. The formation of polymers through disulphide linking causes a viscosity rise 61 in the RVA profile (Lagrain et al., 2005). 62 Most research on the effects of redox agents on gluten has been performed at ambient or 63 proofing temperatures and little attention has been directed to understanding how 64 rheological properties and gluten structure are affected by redox additives during 65 hydrothermal treatment (Attenburrow et al, 1990; Hayta and Schofield, 2004). 66 addition, there do not appear to be any reports describing the combined impacts of redox 67 agents and hydrothermal treatment on gliadin and glutenin separately. Indeed, although In 4 68 redox agents affect the degree of cross-linking of the gluten network (Fitchett and 69 Frazier, 1986) and while it has been shown that dough proteins in the presence of 70 potassium iodate or bromate are less sensitive to heat denaturation (Veraverbeke et al., 71 1999; Yamada and Preston, 1992), their separate impacts on either gliadin or glutenin 72 have, to the best of our knowledge, not been reported. 73 The aim of this study was to further increase insights in the impact of a hydrothermal 74 process on the properties of gluten proteins, and glutenin and gliadin in particular, by the 75 use of redox additives. Hydrothermal treatment was applied using the RVA (Lagrain et 76 al., 2005). The extractability of gliadin and glutenin by SDS, a good indicator of the 77 degree of cross-linking (Hayta and Schofield, 2004), was analyzed at different points 78 during the process using size-exclusion (SE)-high performance liquid chromatography 79 (HPLC). 80 81 5 82 2. Experimental 83 2.1. Materials 84 All reagents were of analytical grade and from Sigma-Aldrich (Steinheim, Germany) 85 unless otherwise specified. Commercial wheat gluten [moisture content: 6.16%, crude 86 protein content (N x 5.7): 78.9% on dry basis (db), starch content: 10.4% db] was from 87 Amylum (Tate & Lyle, Aalst, Belgium) and was used without modification. 88 89 2.2. Controlled heating and cooling 90 The Rapid Visco Analyser (RVA-4D, Newport Scientific, Sydney, Australia) was used to 91 apply temperature profiles to 25.00 g of 20% (w/v) suspensions of control gluten or 92 gluten mixed with different additives. At the start of the RVA analysis suspensions were 93 homogenized by hand-shaking and mixing (900 rpm for 20 s). The temperature profile 94 included 95 1 min), a linear temperature increase to 95 °C in 14 min, a holding step (40 min at 95 °C), 96 a cooling step (7 min) with a linear temperature decrease to 50 °C, and a final holding 97 step at 50 °C (13 min). The RVA was stopped at different points in the heating, holding 98 and cooling phases of the profile and the gluten suspensions frozen in liquid nitrogen, 99 freeze-dried and ground in a laboratory mill (IKA, Staufen, Germany). Potassium 100 bromate [1.52 and 15.2 µmol/g protein (200 and 2000 ppm)], potassium iodate [1.18 and 101 1.77 µmol/g protein (200 and 300 ppm)], and dithiothreitol (DTT) [1.65 and 3.30 µmol/g 102 protein (200 and 400 ppm)] were added as aqueous solutions immediately before RVA 103 analysis. a temperature increase from room temperature to 40 °C (in 6 104 Three characteristic viscosity values were recorded. The initial viscosity, the highest 105 viscosity (in cP) at the start of the RVA run; the minimal viscosity, the lowest viscosity 106 before the holding step; and the maximal viscosity, the highest viscosity in the holding 107 step at 95 °C (Fig. 1). All RVA analyses were performed at least in triplicate. The 108 standard deviations calculated for the initial, the minimal and the maximal viscosities 109 were less than 10% of the respective mean values. 110 111 2.3. Size-exclusion HPLC 112 SE-HPLC was conducted as described by Lagrain et al. (2005). 113 114 2.4. Protein content determination 115 Protein contents were determined as described by Lagrain et al. (2005). 116 117 2.5. Free sulphydryl (SH) determination 118 Free SH groups were determined as described by Lagrain et al. (2005). 119 120 2.6. Statistical analysis of data 121 Significant differences (P < 0.05) in initial, minimal and maximal RVA viscosities were 122 determined by the ANOVA procedure using the SAS package (SAS system for Windows 123 V8, SAS Institute Inc., Cary, NC, USA) and were based on at least three individual 124 measurements. 7 125 3. Results and Discussion 126 The impact of different concentrations of DTT during hydrothermal treatment of a 20% 127 (w/w) gluten-in-water suspension is shown in Fig. 2. As noted earlier (Lagrain et al., 2005), 128 in the control profile, viscosity decreased in the heating step, increased when the gluten- 129 water suspension was held at 95 °C, and decreased rapidly in the cooling phase. Addition of 130 3.30 µmol DTT/g protein (400 ppm) resulted in initial, minimal, and maximal viscosities 131 that were all significantly lower (P < 0.05) than the control viscosities (Table 1). Minimal 132 viscosity was reached at lower temperatures (76.2 °C for 3.30 µmol DTT/g protein) than 133 for the control (91.5 °C) (Fig. 2). The impact of the RVA run on the SDS extractability of 134 the gluten proteins (mainly gliadin) increased with increasing concentrations of DTT (Table 135 2). In the heating step, SDS extractability of gluten proteins with DTT was higher (78.3% at 136 77 °C for 1.65 µmol DTT/g protein) than that of the control gluten (75.7%) (results not 137 shown). However, when gluten suspensions with DTT were held at 95 °C, the extractability 138 rapidly decreased and remained lower than that of the control (Table 3). Similar results (not 139 shown) were obtained when cysteine or glutathione were used instead of DTT. Although no 140 differences in free SH were observed between control gluten and gluten incubated with 141 1.65 µmol DTT/g protein (Table 4), the free SH content in the heating step of a gluten 142 suspension with DTT (7.9±0.3 µmol/g protein at 77 °C for 1.65 µmol DTT/g protein) was 143 higher than that of a control suspension (5.3±0.4 µmol/g protein) and remained higher 144 throughout the RVA analysis (Table 4). These results on the impact of DTT during heating 145 of gluten are generally in agreement with the previously described loss of gluten structure 146 prior to heat setting by reduction of disulphide links, and the subsequent heat induced 8 147 reformation of network structure (Attenburrow et al. 1990) and the decreased extractability 148 in 2.0% SDS (Hayta and Schofield, 2004). 149 Neither potassium bromate (Fig. 3) nor potassium iodate (Fig. 4) had a drastic impact on 150 the initial and minimal viscosity values. However the minimal viscosity was reached at 151 higher temperatures (94.0 °C for 1.18 µmol potassium iodate/g protein and 15.2 µmol 152 potassium bromate/g protein; Table 1). The effect of oxidants was more pronounced in the 153 holding phase at 95 °C where slower viscosity increases were recorded. The gluten-water 154 suspensions with 1.52 µmol potassium bromate/g protein had higher maximal viscosities 155 than the control (Fig. 3, Table 1). With higher concentrations of oxidant, the viscosity 156 increase in the holding step was delayed and was lower than in the control profile. Oxidants 157 increased the protein extractabilities at the end of the RVA run. The effect could be 158 ascribed to higher gliadin extractabilities (Table 2). Whereas the oxidants decreased the 159 SDS extractabilities slightly after 30 min at 40 °C, after 19 min at 95 °C, 1.18 µmol 160 potassium iodate/g of protein resulted in an SDS extractability which was twice that for the 161 heated control sample. At the end of the RVA run, the extractability was almost three times 162 higher. These effects were also observed in the presence of potassium bromate, but to a 163 lesser extent (Table 3). Both oxidants strongly reduced the level of free SH groups of gluten 164 proteins after 30 min at 40 °C (Table 4). The level of free SH of gluten proteins in the 165 presence of oxidants remained lower than that of the control suspension during the holding 166 step at 95 °C. At the end of the RVA analysis, there was no difference in the levels of free 167 SH between gluten suspensions with or without oxidant (Table 4). 168 It has been suggested that oxidants generate bonds between readily accessible thiol groups, 169 and so reduce unfolding of the proteins during heating and decrease interchain disulphide 9 170 bonding in gluten polymers (Attenburrow et al, 1990; Hayta and Schofield, 2004; Nagao et 171 al., 1981). 172 The RVA profiles of gluten-water suspensions supplemented with 1.77 µmol potassium 173 iodate/g protein and 15.2 µmol potassium bromate/g protein were comparable to those of 174 gluten suspensions heated with an SH-blocking agent (100 µmol N-ethylmaleimide/g 175 protein) and to those of control gluten suspensions held at temperatures lower than 95 °C 176 (Lagrain et al., 2005). In addition, protein extractabilities, mainly gliadins, were higher in 177 the presence of oxidants, whereas the free SH content remained lower. The slow RVA 178 viscosity increase in the holding step in the presence of an oxidant indicated lower heat 179 sensitivity as previously observed (Veraverbeke et al., 1999). Oxidants lowered the level of 180 free thiol groups prior to heating (Table 4) and decreased glutenin flexibility. This may 181 hinder sulphydryl-disulphide exchange reactions between gliadin and glutenin that 182 normally occur at temperatures of at least 90 °C (Lagrain et al., 2005; Schofield et al., 183 1983). In agreement with their oxidation potential (Wieser, 2003) the effects were more 184 pronounced for the fast acting oxidant potassium iodate than for the rather more slowly 185 acting oxidant, potassium bromate. In contrast, DTT increased the RVA viscosity at lower 186 temperatures and, although the initial and minimal viscosities were lower due to the action 187 of the reducing agent, the profile almost completely recovered in the holding phase at 95 188 °C. Protein extractabilities in the holding step and at the end of the RVA run were also 189 lower at low concentrations of DTT. This reductant must have increased the heat sensitivity 190 of the gluten proteins and most likely favoured the association of the proteins during 191 thermal treatment through disulphide bonding by initially increasing the molecular 192 flexibility of the glutenin polymers by increasing the levels of free SH-groups that at higher 10 193 temperatures were able to participate in sulphydryl-disulphide interchange reactions leading 194 to an increase in cross-linking of gliadin and glutenin above 75 °C. 195 11 196 4. Conclusions 197 DTT initially lowered RVA viscosity, but still resulted in an RVA viscosity increase at 198 lower temperatures and lower SDS extractabilities than the control suspension. In contrast, 199 potassium iodate and potassium bromate lowered RVA viscosities and increased protein 200 extractabilities in the holding step, suggesting a greater heat resistance of the gluten 201 proteins and less gliadin-glutenin cross-linking. Redox additives may affect the capacity of 202 gluten proteins to associate during heating through sulphydryl-disulphide exchange 203 reactions by altering the level of free thiol groups which can affect the flexibility of 204 glutenin chains and initiate the polymerization reactions. The control of gluten 205 polymerization, and in particular the degree of gliadin-glutenin cross-linking, during 206 processes involving heating, such as bread making and extrusion, may well provide a key to 207 process optimalization. However, further research is necessary to validate the proposed 208 model of hydrothermal treatment of gluten proteins under real processing conditions. 209 210 12 211 Acknowledgements 212 Financial support was obtained from the Institute for the Promotion of Innovation through 213 Science and Technology in Flanders (IWT-Vlaanderen, Brussels, Belgium). Sarah Clarysse 214 is thanked for excellent technical assistance. 215 13 216 References 217 Attenburrow, G., Barnes, D.J., Davies, A.P., Ingman, S.J., 1990. Rheological properties of 218 wheat gluten. Journal of Cereal Science 12, 1-14. 219 Booth, M.R., Bottomly, R.C., Ellis, J.R.S., Malloch, G., Schofield J.D., Timms, M.F., 1980. 220 The effect of heat on gluten-physico-chemical properties and baking quality. Annales de 221 Technologie Agricole 1, 399-408. 222 Fitchett, C.S., Frazier, P.J., 1986. Action of oxidants and other improvers, In: Blanshard, 223 J.M.V., Frazier, P.J. and Galliard, T. (Eds.), Chemistry and Physics of Baking. The Royal 224 Society of Chemistry, London, pp. 179-198. 225 Hayta, M., Schofield, J.D., 2004. Heat and additive induced biochemical transitions in 226 gluten from good and poor breadmaking quality wheats. Journal of Cereal Science 40, 245- 227 256. 228 Lagrain, B., Brijs, K., Veraverbeke, W.S., Delcour, J.A., (2005). The impact of heating and 229 cooling on the physico-chemical properties of wheat gluten-water suspensions. Journal of 230 Cereal Science 42, 327-333. 231 Morel, M.-H., Redl, A., Guilbert, S., 2002. Mechanism of heat and shear mediated 232 aggregation of wheat gluten upon mixing. Biomacromolecules 3, 488-497. 233 Nagao, S., Endo, S., Tanaka, K., 1981. Scanning electron microscopy studies of wheat 234 protein fractions from doughs mixed with oxidants at high temperature. Journal of the 235 Science of Food and Agriculture 32, 235-242. 236 Redl, A., Morel, M.-H., Bonicel, J., Vergnes, B. Guilbert, S., 1999. Extrusion of wheat 237 gluten plasticized with glycerol: Influence of process conditions on flow behavior, 238 rheological properties, and molecular size distribution. Cereal Chemistry 76, 361-370. 14 239 Schofield, J.D., Bottomley, R.C., Timms, M.F., Booth, M.R., 1983. The effect of heat on 240 wheat gluten and the involvement of sulfhydryl-disulfide interchange reactions. Journal of 241 Cereal Science 1, 241-253. 242 Singh, H., MacRitchie F., 2004. Changes in proteins induced by heating gluten dispersions 243 at high temperature. Journal of Cereal Science 39, 297-301. 244 Veraverbeke, W.S., Courtin, C.M., Verbruggen, I.M., Delcour, J.A., 1999. Factors 245 governing levels and composition of the sodium dodecyl sulphate-unextractable glutenin 246 polymers during straight dough breadmaking. Journal of Cereal Science 29, 129-138. 247 Wieser, H., 2003. The use of redox agents, In: Cauvain (Ed.), Bread making, Improving 248 quality. Woodhead Publishing Limited, Cambridge, pp. 424-446. 249 Yamada, Y., Preston, K.R., 1992. Effects of individual oxidants on oven rise and bread 250 properties of Canadian short process bread. Journal of Cereal Science 15, 237-251. 251 15 252 Tables 253 Table 1. Characteristic RVA viscosities (means of at least 3 observations) of gluten-water 254 suspensions (20% w/v) during hydrothermal treatment in the presence of different 255 concentrations of DTT, KBrO3, KIO3. Sample Initial Viscosity (cP) Minimal Viscosity Maximal Viscosity Visc Time T (°C) (cP) (cP) (min) Control 1531a 672b 13.2b 91.2b 2071b C + 1.65 µmol DTT/g protein 1279b 680b 12.8c 89.0c 2020bc C + 3.30 µmol DTT/g protein 1200b 533c 9.6d 76.2d 1523d C + 1.18 µmol KIO3/g protein 1373ab 661b 13.9a 94.0a 1795c C + 1.77 µmol KIO3/g protein 1561a 759a 13.8a 94.0a 1100e C + 1.52 µmol KBrO3/g protein 1306b 650b 13.3ab 91.9b 2383a C + 15.2 µmol KBrO3/g protein 1319b 648b 13.8a 94.0a 1266e 256 Values in the same column followed by a different letter differ significantly (P < 0.05). 257 Relative standard deviations were less than 10% 16 258 Table 2. Extractabilities of gluten proteins in 2.0% SDS before or after RVA analysis in the 259 presence of different concentrations of DTT, KBrO3, KIO3. Sample 260 SDS extractable SDS extractable SDS extractable protein (%) gliadin (%) glutenin (%) Control (before RVA run) 82.4 (0.1) 50.8 (0.4) 31.7 (0.3) Control (C, after RVA run) 16.8 (0.1) 15.7 (0.1) 1.1 (0.1) C + 1.65 µmol DTT/g protein 13.6 (0.2) 12.8 (0.3) 0.9 (0.1) C + 3.30 µmol DTT/g protein 11.1 (0.0) 10.3 (0.0) 0.8 (0.0) C + 1.18 µmol KIO3/g protein 37.0 (1.2) 34.0 (1.4) 3.0 (0.2) C + 1.77 µmol KIO3/g protein 47.9 (2.4) 42.3 (2.1) 5.7 (0.3) C + 1.52 µmol KBrO3/g protein 28.1 (1.1) 26.2 (1.1) 1.9 (0.1) C + 15.2 µmol KBrO3/g protein 56.5 (0.4) 46.2 (0.4) 10.3 (0.0) Standard deviations are shown in brackets. 17 261 Table 3. Extractabilities of gluten proteins in 2.0% SDS with addition of DTT, KBrO3, 262 KIO3 during RVA analysis. Total SDS extractability (%) RVA Temperature (°C) 1,65 µmol 1.18 µmol 1.52 µmol DTT/g protein KIO3/g protein KBrO3/g protein Control 401 82.4 (0.1) 82.5 (2.6) 77.2 (0.3) 79.2 (1.2) 952 24.8 (1.0) 19.0 (1.8) 50.9 (2.8) 40.3 (0.8) 503 18.9 (0.5) 14.4 (0.0) 37.0 (1.2) 30.9 (0.4) End 16.8 (0.1) 13.6 (0.2) 37.0 (1.2) 28.1 (1.1) 263 1 After 30 min at this temperature 264 2 After 19 min at this temperature 265 3 In the cooling step 266 Standard deviations are shown in brackets. 267 18 268 Table 4. Free SH content (µmol SH/g protein) of gluten proteins with addition of DTT, 269 KBrO3, KIO3 during RVA analysis as determined by the reaction with DTNB in 2% (w/v) 270 SDS, 3.0 M urea, 1.0 mM EDTA, 0.05 M NaH2PO3. RVA Temperature (°C) 1,65 µmol 1.18 µmol 1.52 µmol DTT/g protein KIO3/g protein KBrO3/g protein Control 401 7.6 (0.2) 7.5 (0.0) 3.5 (0.2) 4.7 (0.0) 952 2.1 (0.1) 2.6 (0.1) 1.6 (0.1) 1.6 (0.2) 503 2.5 (0.1) 2.7 (0.1) 2.3 (0.2) 2.2 (0.1) End 2.6 (0.5) 3.1 (0.7) 2.5 (0.4) 2.5 (0.6) 271 1 After 30 min at this temperature 272 2 After 19 min at this temperature 273 3 In the cooling step 274 Standard deviations are shown in brackets. 19 275 Fig. captions 276 Fig. 1. RVA profile (40 min HT at 95 °C) of a gluten-water suspension with indication of 277 characteristic viscosity values. Initial viscosity (A), minimal viscosity (B), maximal 278 viscosity (C). 279 Fig. 2. RVA profiles (40 min HT at 95 °C) of gluten-water suspensions with DTT. Control 280 viscosity profile ( 281 ( 282 Fig. 3. RVA profiles (40 min HT at 95 °C) of gluten-water suspensions with potassium 283 bromate. Control viscosity profile ( 284 KBrO3/g protein( 285 Fig. 4. RVA profiles (40 min HT at 95 °C) of gluten-water suspensions with potassium 286 iodate. Control viscosity profile ( 287 KIO3/g protein ( ), temperature ( ), 1.65 µmol DTT/g of protein ( ), 3.30 µmol DTT/g protein ). ), temperature ( ), temperature ( ), 1.52 µmol KBrO3/g protein( ), 15.2 µmol ). ), 1.18 µmol KIO3/g protein ( ), 1.77 µmol ). 288 289 20 290 Fig.s 291 Fig. 1 C 100 A Viscosity (cP) 2000 80 B 1500 60 1000 40 500 20 0 0 0 292 Temperature (°C) 2500 10 20 30 40 50 60 70 Time (min) 21 2500 100 2000 80 1500 60 1000 40 500 20 0 0 0 294 Temperature (°C) Fig. 2 Viscosity (cP) 293 10 20 30 40 50 60 70 Time (min) 22 295 Fig. 3 2500 100 2000 80 1500 60 1000 40 500 20 0 Temperature (°C) Viscosity (cP) 296 0 0 10 20 30 40 50 60 70 Time (min) 297 298 23 299 Fig. 4 2500 100 2000 80 1500 60 1000 40 500 20 0 0 0 301 Temperature (°C) Viscosity (cP) 300 10 20 30 40 50 60 70 Time (min) 24