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Wheat flour superheated steam treatment can improve the baking food quality by modifying the functionality of wheat flour. To reveal the underlying mechanism of improving wheat flour quality by superheated steam treatment, the gluten molecular rearrangement and polymerization behavior of wheat flour upon superheated steam were studied. Molecular size distributions of samples were analyzed using size exclusion (SE-HPLC) and electrophoresis, which illustrated that superheated steam treatment promoted the aggregation of gluten due to cross-links of disulfide (SS) bonds. Superheated steam treatment increased the SDS-insoluble proteins content from 63.07% (native wheat flour) to 93.32% (treated at 150 °C for 1 min). Infrared spectroscopy (FTIR) was used to calculate the different secondary structures percentages of gluten, suggesting that superheated steam treatment increased intermolecular β-sheet and α-helix while it decreased anti-parallel β-sheet, β-turn and β-sheet. Furthermore, thermo gravimetric analysis (TGA) and rheology suggested that superheated steam treatment enhanced gluten mass but weakened gel system due to molecular rearrangements of SS bonds and hydrogen bonds. [Display omitted] • Gluten polymerization behavior of wheat flour upon superheated steam were studied. • Superheated steam treatment induced gluten aggregation and SS bonds cross-links. • Superheated steam treatment weakened the wheat flour gel system. [ABSTRACT FROM AUTHOR]