Rice; starch; RVA; amylopectin; digestibility.

Various factors, including starch granule channels, have been suggested to contribute to the control of sorghum starch digestibility for animal feed. Isolated starch from two normal sorghum lines (P721N, IS6986) and one high protein digestibility (HPD) mutant line (111) that differed in starch granule morphology were selected to study the influence of these factors on starch digestibility. Scanning electron micrographs were taken of raw and digested starches. Microscopy results confirmed that in all three sorghum lines channels in starch granules are the main route of enzyme penetration and the central cavity area is the starting point of enzyme digestion. Channel density was more pronounced in the HPD sorghum mutant line than in normal lines, which may have been responsible for its relatively high digestibility. Micrographs of IS6986 showed unique starch granule morphology with a collapsed "doughnut-shaped" structure in a portion of the granules. These unusual granules were rapidly digested and, unlike normal spherical granules, totally disappeared after 30 min of digestion. Amylases appeared to have fast access to the collapsed-appearing starch granules. Digestion profiles, following incubation with pepsin and a-amylase, showed that IS6986 and the HPD mutant (111) had the highest initial rate of starch digestion, followed by P721N. These findings provide insight as to how new sorghum cultivars might be developed with high starch digestibility for animal feed use.

Proteins were detected in channels of commercial starches of normal maize, waxy maize, sorghum, and wheat through labeling with a protein‐specific dye and examination using confocal laser scanning microscopy (CLSM). The dye, specifically 3‐(4‐carboxybenzoyl)quinoline‐2‐carboxaldehyde (CBQCA), fluoresces only after it reacts with primary amines in proteins, and CLSM detects fluorescence‐labeled protein distribution in an optical section of a starch granule while it is still in an intact state. Starch granules in thin sections of maize kernels also had channel proteins, indicating that proteins are native to the channels and not artifacts of isolation. Incubation of maize starch with protease (thermolysin) removed channel proteins, showing that channels are open to the external environment. SDS‐PAGE analysis of total protein from gelatinized commercial waxy maize starch revealed two major proteins of about Mr 38,000 and 40,000, both of which disappeared after thermolysin digestion of raw starch. Commercial waxy maize starch granule surface and channel proteins were extracted by SDS‐PAGE sample buffer without gelatinization of the granules. The major Mr 40,000 band was identified by MALDI‐TOF‐MS and N‐terminal sequence analysis as brittle‐1 (bt1) protein.

Using the small intestine enterocyte Caco-2 cell model, sucrase-isomaltase (SI, the mucosal α-glucosidase complex) expression and modification were examined relative to exposure to different mono- and disaccharide glycemic carbohydrates. Caco-2/TC7 cells were grown on porous supports to post-confluence for complete differentiation, and dietary carbohydrate molecules of glucose, sucrose (disaccharide of glucose and fructose), maltose (disaccharide of two glucoses α-1,4 linked), and isomaltose (disaccharide of two glucoses α-1,6 linked) were used to treat the cells. qRT-PCR results showed that all the carbohydrate molecules induced the expression of the SI gene, though maltose (and isomaltose) showed an incremental increase in mRNA levels over time that glucose did not. Western blot analysis of the SI protein revealed that only maltose treatment induced a higher molecular weight band (Mw ~245 kDa), also at higher expression level, suggesting post-translational processing of SI, and more importantly a sensing of maltose. Further work is warranted regarding this putative sensing response as a potential control point for starch digestion and glucose generation in the small intestine.

The proposed rapid small‐scale starch isolation technique in the laboratory was a combination of dry grinding of grain, suspension of the resulting flour in extraction buffer, application of ultrasonic sonication, then separation by sucrose density centrifugation. Light microscopy of separated fractions showed intact starch granules in the pellet and proteins and damaged starch in the top layer. The extraction method yielded 61% starch from sorghum and 63% from maize. The isolated starch showed lower starch damage and proteins content than by the conventional method. The gelatinization enthalpy of isolated starch was slightly higher than by wet grinding conventional method. In addition to low amount of starting flour (100 mg) the new starch isolation method was performed in less than 2 h from dry grinded seed to dried starch. Thus, it could be a useful method for cereal chemists and plant genetists.