Analyzing the starch molecular structure

Research on starch has lasted for two centuries, and it is amazing to find that a final consensus about its molecular structure has not been achieved yet. The same is true with regard to the structure of the starch granules.

Characterizing structures of starch components

Starch is a type of carbohydrate made of two main components: amylose and amylopectin. Amylopectin is the major component and is made of many short chains connected by (1->6) linkages, making it extensively branched. Amylose consists of either a single or a few long chains and is 20-30% of most starches. The shape of high-amylose starches granules is often deformed and they may contain an additional component called intermediate material. The structure of starch components is difficult to analyze because of its simple chemical composition. Parameters like the degree of polymerization (DP) are used to characterize starch components, especially amylopectin. The macromolecule contains A, B, and C chains, which are divided into long and short chains and segments like external, total internal, and internal chains. There are two structural models to explain the arrangement of the chains in amylopectin, but there’s no consensus on which model is more likely valid.

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Fractionation of starch

The fractionation of starch is a process to separate the components amylose, amylopectin, and IM. Starch samples are first cleaned of fatty material, then dissolved in dimethyl sulfoxide (DMSO), 6-10 M urea solution, or KOH/NaOH. Dissolved starch is then precipitated and redissolved in ethanol or fractionated using classical methods with n-butanol, a commercial mixture of amyl alcohols, or a thymol complex. Another method uses the protein lectin concanavalin A to precipitate amylopectin. Gel-permeation chromatography (GPC) can also be used for small-scale fractionation, where amylopectin is eluted at the void volume and amylose is partly included in gel particles.

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Analysis of amylose

Though amylose is a minor component in most granules, it has a large influence on the properties of starch. The traditional definition of amylose is a molecule composed of a long, linear chain of (1/4)-linked α-D-glucosyl units. Most starch preparations contain, however, also slightly branched amylose molecules. There is no specified smaller size for the definition of amylose. In some cases, even comparatively short linear dextrins, obtained by hydrolysis of amylopectin, were called amylose. This is unfortunate because it tends to be a source of confusion. Chains of lengths comparable to the short chains of amylopectin should preferentially be called linear oligosaccharides.

Amylose Content of Starch

The first proposed helical structure for biopolymer was for amylose (Hanes, 1937). The blue complex with iodine was later confirmed to involve amylose in a helical conformation (Rundle and French, 1943). The color and intensity of the complex depend on the chain length (CL) of amylose. Shorter chains have a red color while longer chains have a blue color. The blue value (BV) is defined as the absorbance of 1 mg of starch in 100 mL of a mixture containing iodine and potassium iodide, with BV for amylose ranging from 1.01-1.63 and for amylopectin ranging from 0.08-0.38. BV is a qualitative test for amylose, but should not be considered quantitative.

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Structural Analysis of Amylose

The size of amylose molecules is measured as the Degree of Polymerization (DP) using light scattering, reducing end analysis with the Parke-Johnson reagent, or limiting viscosity number. The DP varies between 0.51 and 6.34×103 across different plants. The size distribution was analyzed using HPSEC with simultaneous refractive index and low-angle laser light scattering detection. The size distribution for amylose from different sources showed a broad distribution for potato and tapioca, but a more narrow distribution for kudzu. Multi-angle light scattering detectors have replaced LALLS for molecular weight estimation.

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Analysis of amylopectin structure

The molecular weight of amylopectin is larger than that of amylose and presents challenges in obtaining accurate average estimations due to its tendency to form molecular aggregates and the risk of fragmentation. The molecular weight of amylopectin can range between 2 and 700 million, depending on the source of the plant, method of determination, and solvent used. Flow field-flow fractionation (FFFF) has become an alternative method to traditional methods such as GPC and SEC for the separation of starch components and has the advantage of minimizing the risk of sample fragmentation and providing a practically unlimited size-fractionation range. The Mw values for amylopectin obtained by FFFF range between 0.45 and 4.5 billion g/mol, depending on the source of the plant.

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Analysis of intermediate materials (IM)

A starch component called “IM” (intermediate material) found in some high-amylose starches. IM has gained attention for its potential contribution to the fraction of resistant starch, which is thought to have health benefits. IM has been found in several starches including maize, potato, barley, and oats, among others, but its nature can vary and might not be known in some cases. IM can interfere with the measurement of amylose content by iodine binding, leading to overestimation. IM is structurally related to either amylose or amylopectin and is analyzed using the same methods.

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