1. Enoch T. Quayson 1,a, Alessandra Marti 1,2 and Koushik Seetharaman 1 1 Department of Food Science and Nutrition, University of Minnesota, Saint Paul, MN, US 2 Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milan, Italy a quays001@umn.edu 3) MATERIALS  Commercial Hard Wheat Flour (HWF) – 12% protein  Commercial Soft Wheat Flour (SWF) - 8% protein kindly provided by Horizon Milling (Mankato, MN, US) 2) AIM Investigating the effect of mixing temperature on protein secondary structure and thiol content in hard and soft wheat flours * In memory of Dr. Koushik Seetharaman Effect of mixing temperature on protein secondary structure and thiols in soft and hard wheat flour dough* 1) BACKGROUND  Dough rheology is important in the manufacture of cereal-based products  Differences in dough rheology have primarily been attributed to differences in protein quantity and quality of the flours  Various studies have examined the importance of temperature on gluten quality characteristics (Rakita et al., 2014; Stathopoulos et al., 2008). However, they often involved extraction of gluten and its subsequent heating within the 25-95°C temperature range  There is dearth of information on dough protein characteristics at temperatures below 25°C 4) EXPERIMENTAL Dough preparation  Farinograph-AT (C.W. Brabender Inc., South Hackensack, NJ, US)  AACC method (54-21) for the identification of optimal water absorption to reach the 500 Farinograph Units (FU) Dough sampling  Dough Development Time (min): time from first addition of water to the point of maximum consistency range  Mid-Stability (MS): half time (min) between stability arrival (time point where top of curve first intersects 500 FU line) and stability departure  Time to Breakdown (min): time from start of mixing until there has been decrease of 30 FU from peak points  Stability Departure (min): time point where top of curve leaves 500 FU line Attenuated Total Reflectance (ATR) – Fourier transform infrared (FTIR) spectroscopy  Bruker Tensor 37 (Bruker Optics, Inc., Billerica, MA, USA)  Spectra aquisition: spectral range: 4000-600 cm -1 ; 32 scans at 4 cm -1 resolution  Specta elaboration (Bock and Damodaran, 2013 : spectra normalisation; H 2 O/D 2 O spectra subtraction; second derivative; integration Estimation of protein secondary structure -sheet: 1620-1644 cm -1 ; Random: 1644-1652 cm -1 ; -helix: 1652-1660 cm -1 ; -turn: 1660-1685 cm -1 Dough lyophilization  Accessible Thiols 0.5mM 5,5’-dithiobis(2-nitrobenzoate) (DTNB) assay (Iametti et al., 2006)  Total Thiols DTNB assay in presence of 1% sodium dodecyl sulfate (SDS) (Iametti et al., 2006) Figure 1: Mixing profile of hard (a) and soft (b) wheat flours at different temperatures Table 1. Farinograph indices Soft Wheat (SWF) Hard Wheat (HWF) 4°C15°C30°C40°C4°C15°C30°C40°C Water Absorption (g/100g flour)68.761.555.552.381.474.264.761.6 Dough Development Time (min:ss) 09:4005:0601:01 16:4812:3302:4401:59 Mid-Stability (min:ss)10:3306:2201:2801:1720:1814:1509:5203:00 Stability Departure (min:ss)15:5411:2202:1601:4627:4119:2716:3305:10 Time to Breakdown (min:ss)14:1109:2401:5401:4824:4216:4816:0004:42 Stability (min:ss)10:4210:4101:3301:0215:5313:1915:0404:05 (b)(a)  For both SWF and HWF, the water absorption decreases as the temperature increases  Dough development time and stability decrease as mixing temperature increases, indicating that at high temperature the dough required less energy for developing  Mixing of SWF at 15°C leads to strong dough, with a stability similar to that of HWF  Mixing of HWF at 15°C shows dough stability comparable with that of the dough mixed at 30°C 5) RESULTS: MIXING PROPERTIES 6) RESULTS: PROTEIN CONFORMATION Figure 2. Estimation of protein secondary structure of hard (a, c) and soft (b, d) wheat dough at the dough development time (a,b) and stability departure (c, d)  Regardless of the type of flour, β-sheets > β-turns>random>-helix  In SWF, β-sheets increase at the expenses of β-turns as temperature increases  HWF dough show secondary structural “stability” as temperature increases and mixing progresses  Differences in secondary structural changes for HWF and SWF may explain the differences in dough strengths between the two flours 7) RESULTS: ACCESSIBLE AND TOTAL THIOLS  Hard and soft wheat doughs have comparable amounts of accessible thiols  Soft wheat dough has higher total thiols than hard wheat dough  Accessible thiols in soft wheat dough are comparable for all mixing points and temperatures  Lowest value of accessible thiols was observed in hard wheat dough at 15°C at the dough development time and at 30°C at the stability departure point  Total thiols in hard wheat dough decreases as temperature increases, suggesting a thiol- disulfide interchange as temperature increases  Soft wheat dough at 30°C showed higher total thiols than 15°C, likely indicating thiol – disulfide interchange at low temperature  No change in total thiols is observed between 15°C and 40°C for soft wheat dough  Temperature is more important than mixing in the thiol-disulfide interchange in HWF evident by the insignificant difference in total thiols between dough development time and stability departure for all temperatures 8) CONCLUSIONS  Soft and hard wheat dough have different changes in secondary structure and thiols for different mixing times and temperatures  β-sheets and β-turns are more stable in hard wheat dough than in soft dough as temperature increases  Mixing in the 4-15°C range represents the best solution for soft wheat dough in order to have a β-sheets/β-turns ratio similar to that of hard wheat dough at 30°C and to increase S-S bonds REFERENCES Figure 3. 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