Determination of protein secondary structure in wheat flour-water systems during mixing using Fourier transform horizontal attenuated total reflectance infrared spectroscopy

For the first time, an infrared spectroscopic method has been developed to determine changes in the secondary structure of gluten proteins in a flout-water dough system as it is mixed. Fourier transform horizontal attenuated total reflectance (FT-HATR) spectra of mixed doughs were collected in the mid-IR (4000–700 cm−1), emphasizing the amide III spectral region (1350–1200 cm−1). FT-HATR spectra of incrementally mixed doughs revealed four bands in the amide III region typically associated with secondary structure of proteins: they include 1317 (α-helix), 1285 (β-turn), 1265 (random coil), and 1242 cm −1 (β-sheet). A fifth band at 1339 cm−1 has been tentatively assigned as α-helix. The two largest bands, which also showed the greatest change in second derivative band area (SDBA) during mixing, were the bands at 1339 cm−1 (decreasing over time) and 1242 cm−1 (increasing over time). Correlation of the ratio of the SDBA at 1339 cm−1 over 1242 cm −1 and the SDBA at 1242 cm−1 to the mixogram midline point, were 0.88 and 0.97, respectively. The bands at 1317, 1285, and 1242 cm−1 also showed increases in SDBA's over time to an optimum, which had high correlations to the mixogram midline point. On the other hand, the bands at 1339 and 1265 cm−1 showed a corresponding decrease as the doughs were mixed, and also had high correlations to the mixogram midline point. These observations suggest that as flour-water dough systems progress through hydration, development, and then breakdown during the mixing process, the secondary structure of gluten protein assumes a more ordered conformation as seen by increases in α-helical, β-turn, and β-sheet structure, and do so at the expense of random coil structure in the gluten macromolecule. Since hydrogen bonding significantly stabilizes all of these new conformations, this implies that hydrogen bonding within the gluten macromolecule also increases during mixing. These results demonstrate that it is possible to accurately and objectively monitor the rheological behavior of dough systems based on changes in the protein structure of the system without the need for traditional and more subjective systems that simply measure the physical response of the dough to work input.