Molecular Fractionation of Starch in Nycodenz-Dimethyl Sulfoxide by Density-Gradient Ultracentrifugation

Abstract

Starches from various origins (normal corn, waxy corn, high-amylose corn, normal rice, waxy rice, potato and tapioca) were fractionated by density-gradient ultracentrifugation at 124,000 × g, using a gradient of Nycodenz (0-40%, w/w) in 90% dimethyl sulfoxide (DMSO). The fractions of different densities were collected from a centrifuge tube, and then analyzed by the phenol-sulfuric acid method and iodine-binding test. Amylose and amylopectin were clearly separated by the density-gradient centrifugation within 6 h of centrifugation in the total carbohydrate vs. density profile. The amylose content in the starches, measured from the centrifugation for 6 h, agreed with the values reported in the literature, and the content of intermediate fraction (5-13%) was also measured. Amylopectin quickly reached a density region (1.22-1.23 g/mL) with a sharp and stable peak, whereas amylose showed a band in a broad density range, moving toward higher density as the centrifugation continued. The buoyant density of amylose became similar to that of amylopectin after extensive centrifugation. Because the sedimentation rate of amylose was much lower than that of amylopectin, both starch chains are effectively fractionated in the gradient medium, and the iodine-binding property of the fractionated starches provides valuable information on the chain conformation of starch.