Effects of salt on the formation of gluten network upon hydration Poster Title: Font size and line spacing can be altered in this section to suit the length of your title. Your title must be vertically aligned to the bottom. H.C.D.Tuhumury123, D. M. Small2, L. Day1 Author names and affiliation: This information must always be placed above the ribbon below the title and subtitle. If more space is required, affiliations can be listed below the bottom ribbon where the references and acknowledgements are placed. 1CSIRO Animal, Food and Health Sciences, Werribee, VIC 3030, Australia 2 School of Applied Sciences, RMIT University, VIC 3001, Australia 3Faculty of Agriculture, Pattimura University, Ambon, 97233, Indonesia CSIRO Animal, Food and Health Sciences Introduction Salt (NaCl) plays an important technological role in the processing of wheat flour based foods. Salt affects the gluten protein network formed during the mixing of flour with water, which in turn, influences the dough strength and dough-handling properties. However, effects of salts on gluten protein network during mixing at the molecular level particularly as these relate to rheological properties are still not well understood. Our hypothesis is that salt affects the formation of the gluten network at the very first initial hydration of the gluten proteins during mixing, which affects the non-covalent interactions between the gluten proteins hence its rheological properties . A1 A2 C1 C2 Materials and methods 25 μm 25 μm 25 μm 25 μm Materials Flour : FSB and Redbase (13.2 and 10.4% protein) Water-washed (WW) gluten obtained by mixing the flour with water (60% moisture) and washing the dough in water Salt-washed (SW) gluten obtained by mixing the flour with 2% NaCl (flour base) and washing the dough in salt solutions B1 B2 D1 D2 Methods Rhelogical measurement: Strain sweep at linear viscoelastic region (0.05%-10% strain); frequency 1 Hz; 25°C using a controlled stress rheometer (Paar Physica MCR 300) Microscopic analysis : Confocal laser scanning microscope (CLSM, Leica SP5) and transmission electron microscope (TEM, JEOL 1000) Biochemical analysis : Size-exclusion HPLC (Glu:Gli ratio, proportion of unxetractable polymeric protein (% UPP); Disulfide-sulfhdryl analysis. Secondary structure analysis : ATR- FTIR spectroscopy (Perkin Elmer Spotlight 400) 128 scans at 4 cm-1 in the range of 650-4000 cm-1. . 5 μm 5 μm 5 μm 5 μm Figure 2: Microstructure of rehydrated gluten (A-confocal; B-TEM) and fresh gluten (C-confocal, D- TEM): Water washed (1) and salt-washed (2) Salt causes the gluten proteins to form fibrous structure compared to that of without salt which have network consisting of large protein aggregates (Fig. 2) Similar network structure was encountered in the freshly prepared gluten i.e. fibrous structure and large aggregates for gluten mixed with salt and without salt, respectively. These results indicate that in the present of salt the gluten proteins form more ordered alignment resulting in fibrous structure. This structure was formed during the initial protein hydration and dough mixing, since the structure was maintained upon rehydration. 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Results and discussion Table 1: Biochemical properties of the WW and SW gluten All gluten samples have storage modulus G’ values higher than the loss modulus G’’, indicating that the gluten has viscoelastic solid-like behaviour (Fig. 1) Glutens obtained with salt from both flours have higher tan δ values (less elastic) compared to those without salt The gluten structural network formed in the presence of salt during initial hydration and subsequent washing resulted in less elastic behaviour not only for the rehydrated gluten but also the fresh gluten The degree of the difference in the viscoelasticity (tan σ) of the gluten network as affected by salt appears to be determined by the gluten from the individual flour Sample Glu:Gli ratio of SDS-extractable protein Glu:Gli ratio of total protein % UPP Free SH content* Total SH content* Disulfide content* WWFSB 0.45 ± 0.01 b 0.79 ± 0.05 a 58.6 ± 7.17 a 3.5 ± 0.25 b 49.2 ± 3.29 b 23.0 ± 1.69 b SWFSB 0.38 ± 0.01 c 0.73 ± 0.01 a 61.5 ± 5.32 a 2.3 ± 0.10 c 48.3 ± 1.54 b 23.0 ± 0.77 b WWRedbase 0.49 ± 0.01 a 0.82 ± 0.02 a 53.9 ± 0.79 a 5.3 ± 1.41 a 63.1 ± 4.40 b 28.9 ± 2.07 b SWRedbase 0.43 ± 0.01 b 0.79 ± 0.01 a 58.1 ± 0.01 a 2.6 ± 0.47bc 61.7 ± 2.49 a 29.6 ± 1.35 a Means in the column with same letter are not significantly different (P≤ 0.01) a Unextractable polymeric protein; * nMoles/mg protein The presence of salt during mixing and washing of the dough resulted in the gluten having no differences in Glu:Gli ratio of total protein extract, % UPP, and disulfide content but significant differences in the Glu:Gli ratio of the SDS-extractable protein extract and free SH content to gluten prepared without salt (Table 1) These results suggested that the different rheological properties of the gluten as affected by salt were not due to covalent interactions between polymeric glutenins protein through disulfide linkage, but through the non-covalent interactions within the gluten structure Table 2: Peak possition and relative areas of bands fitted to deconvoluted spectra of WW and SW gluten Band assignment and peak centre Relative peak area (%) WW FSB SW FSB WW Redbase SW Redbase β-sheet (1612, 1620, 1630, 1681) 45.9 ± 0.22 45.7 ± 0.32 48.1 ± 0.59 58.0 ± 2.14 α-helix (1650) 37.7 ± 5.73 41.4 ± 0.44 36.1 ± 0.52 31.0 ± 0.01 β-turn (1670) 16.4 ± 5.95 12.9 ± 0.19 15.8 ± 0.07 11.0 ± 2.14 With addition of salt, an increase in the intermolecular β-sheet structure of the gluten was obviously observed in the gluten prepared from Redbase flour but remained the same for the gluten from FSB flour. he and a decrease in β-turn from both flour. (Table 2) These results confirmed that salt cause conformational changes in the secondary structure of the gluten during hydration, where hydrogen bond and hydrophobic interactions play a dominant role. Figure 1: Rheological properties of the rehydrated and freshly prepared water-washed (WW) and salt-washed (SW) gluten at small strain. Conclusion The effect of salt on gluten network formation is during the initial hydration of the flour (early stages of the mixing). Once the gluten is formed, rehydration with water does not change its structure. Although the presence of salt during dough mixing did not influence covalent interactions between glutenins to form large polymer network, it is likely that salt changes the solvent quality of water which affects hydrogen bonding and hydrophobic interactions, hence resulted increased β-sheet structure in the gluten and allow the protein molecules to be better aligned to form fibrous structure with higher tan δ values thereby less elastic network. Further information: Insert your contact details, including a divisional/ unit specific url. REFERENCES BELTON, P. S. 1999. Journal of Cereal Science, 29, 103-107. CHAN, K.-Y. & WASSERMAN, B. P. 1993. Cereal Chem., 70, 22-26 FU, B. X., SAPIRSTEIN, H. D. & BUSHUK, W. 1996. Journal of Cereal Science, 24, 241-246 GUPTA, R. B., KHAN, K. & MACRITCHIE, F. 1993. Journal of Cereal Science, 18, 23-41 . ACKNOWLEDGEMENTS The authors acknowledge the AusAid for the Australian Development Scholarship to Ms Helen Tuhumury The authors acknowledge the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility at the RMIT Microscopy & Microanalysis Facility, at RMIT University. KINSELLA, J. E. & HALE, M. L. 1984. Journal of Agricultural and Food Chemistry, 32, 1054-1056. McCANN, T. H., SMALL, D. M., BATEY, I. L., WRIGLEY, C. W. & DAY, L. 2009. Food Chemistry, 115, 105-112 UKAI, T., MATSUMURA, Y. & URADE, R. 2008. Journal of Agricultural and Food Chemistry, 56, 1122-1130.. Li Day e Li.Day@csiro.au w www.csiro.au/ CAFHS References & acknowledgements: Can appear below the bottom ribbon if you don’t have enough room on your poster.