Molecular fractionation of starch by density-gradient ultracentrifugation

Abstract

Amylose and amylopectin in corn and potato starches were fractionated by centrifugation at 124,000 g for 3–72 h at 40 °C in a gradient media, Nycodenz, based on their sedimentation rate differences. The fractions were collected from a centrifuge tube, and then analyzed by the phenol–sulfuric acid method and iodine-binding test. Amylopectin, a large and highly branched starch molecule, migrated faster than amylose and quickly reached its isopycnic point with a buoyant density of about 1.25 g/mL, exhibiting a sharp and stable carbohydrate peak. Amylose, which is a relatively small and linear molecule, however, migrated slowly in a broad density range and continued moving to higher density regions, eventually overlapping with amylopectin peak as the centrifugation continued. This could indicate that the buoyant density of amylose is similar to that of amylopectin. Under centrifugal conditions of 3 h and 124,000 g, amylose and amylopectin molecules were clearly separated, and the presence of intermediate starch molecules (11.5 and 7.7% for corn and potato starch, respectively) was also observed between amylose and amylopectin fractions. The amylose content of corn and potato starches was 22.6 and 21.1%, respectively, based on the total carbohydrate analysis after the ultracentrifugation for 3 h. In alkaline gradients (pH 11 or 12.5), the sedimentation rate of starch molecules and the buoyant density of amylopectin were reduced, possibly due to the structural changes induced by alkali.