We investigated the effects of salt addition (0%, 1%, 2%, 3%) and homogenization speed (4000, 6000, 8000, 10,000 rpm) on the yield and aggregation characteristics of gluten fractions separated through a laboratory-scale batter procedure. The gluten yield was linearly correlated with the A-starch yield and increased with salt concentration and the homogenization rate. Adding salt and increasing the homogenization rate reduced the surface hydrophobicity and enhanced the hydration of gluten. The difference in the water-binding capacity of gluten, A- and B-starch granules is the basis for the centrifugal separation. The increase in gluten water-binding capacity promotes the separation of the three phases. Meanwhile, hydration shifted the secondary structure of gluten to β-turn, which enhanced its tensile resistance. In the presence of salt and mechanical forces, free sulfhydryl groups undergo cross-linking reactions to generate disulfide bonds, leading to the polymerization of glutenin macropolymer and the increase of gliadin in SDS-extractable proteins. [Display omitted] • Batter was prepared with different levels of salt (0–3%). • Batter was homogenized with four speed levels (4000–10000 rpm). • Salt and high homogenization speed significantly increase the yield of gluten and starch. • Salt and high homogenization speed enhanced the macromolecular aggregation of gluten. • Gluten with higher levels of salt and homogenized speed exhibited greater strength. [ABSTRACT FROM AUTHOR]