Amylopectin determination

Table 10.8 Chain-length distributions of amylopectins determined after debranching3...

Ratios of A- and B-Chains in Samples of Glycogen and Amylopectin, Determined from Measurements of c.L, beto-Amylolysis Limits, and Fhosphorolysis Limits ... 

Rice is one of the world s primary food crops and is a major source of dietary energy for more than half of the people on this planet. Paddy is usually milled or polished into white rice for cooking even though unpolished rice contains much more nutritional bioactive components and dietary energy. A major component of rice grain is starch, which in turn is composed of polysaccharides two types of polysaccharides found in rice starch are amylose and amylopectin. The amounts of amylose and amylopectin determine the quality of rice, both in terms of physical and chemical characteristics. 

A 0.2-g sample of amylopectin was analyzed to determine the fraction of the total glucose residues that are branch points in the structure. The sample was exhaustively methylated and then digested, yielding 50 /tmol of 2,3-dimethylglucose and 0.4 /tmol of 1, 2, 3, 6-tetramethylglucose. 

Molecular Structure. Most starches consist of a mixture of two polysaccharide types amylose, an essentially linear polymer, and amylopectin, a highly branched polymer. The relative amounts of these starch fractions in a particular starch are a major factor in determining the properties of that starch. 

It is obviously important that the fractionation products should be adequately characterized. The only accurate method for ascertaining the purity of the starch components, and also the amylose/amylopectin ratio in whole starch, is to determine potentiometrically the amount of iodine bound.8 38 Colorimetric methods which have been suggested37 38 are useful for comparative measurements, but are often not absolute. The yield of... 

Methods which can be used to determine the size and shape of polysaccharides have been reviewed.107 (A critical survey of these has recently been given by Sadron108 and by Ogston.109) Special problems exist in the case of the undegraded starch components. In view of the branched nature of amylopectin and the large size of the amylose molecule, chemical methods of estimating size are inadequate, and it is questionable whether results are valid.38 The free components may also aggregate in aqueous solution. Study of derivatives is therefore more convenient, and the preparation of these is an essential preliminary to estimations of molecular size. 

The Results of Molecular-Weight Determinations on Amylopectin and its Derivaiives... 

Amylo-1 —> 6-glucosidase obtained by Cori and Larner218 from rabbit muscles, and R-enzyme isolated by Hobson, Whelan and Peat219 from potatoes and broad beans, are typical debranching enzymes, which will hydrolyze the 6 — 1-a-D-glucosidic linkage rather than the normal 4 —> 1-a-D linkage. These enzymes will therefore be particularly important in determinations of the fine structure of amylopectin, if they can be sufficiently well purified. 

Pea starch granules are oval, sometimes fissured, with a diameter of 20-40 ym (13). Molecular and structural characteristics of the two main components of field pea starch—amylose and amylopectin—are important in determining functional properties (25,26). Smooth field pea starch concentrate contains 97.2% starch of which 30.3-37.8% is amylose (9,23,25-27), and wrinkled pea starch concentrate contains 94.8% starch, which is 64% amylose (26). The gelatinization temperature of smooth pea starch is between 64 to 69 C, and that of wrinkled pea starch is greater than 99 C to 115 C. Gelatinization temperature depends on maturity of field pea seed and amylose content (26,27). 

To determine the amylose content of starch, the iodine reaction has been most commonly used because amylose and amylopectin have different abilities to bind iodine. The methods such as blue value (absorbance at 680 nm for starch-iodine complex using amylose and amylopectin standards), and potentiometric and amperometric titration have been used for more than 50 years. These procedures are based on the capacity of amylose to form helical inclusion complexes with iodine, which display a blue color characterized by a maximum absorption wavelength (kmax) above 620 nm. During the titration of starch with iodine solution, the amount (mg) of iodine bound to 100 mg of starch is determined. The value is defined as iodine-binding capacity or iodine affinity (lA). The amylose content is based on the iodine affinity of starch vs. purified linear fraction from the standard 100 mg pure linear amylose fraction has an iodine affinity of 19.5-21.0mg depending on amylose source. Amylopectin binds 0-1.2mg iodine per 100mg (Banks and Greenwood, 1975). The amylose content determined by potentiometric titration is considered an absolute amylose content if the sample is defatted before analysis. 

Starch (20 mg, dry basis) in water (10 mL) is heated at certain temperatures in sealed tubes for 30 minutes. The tubes are then cooled to room temperature and centrifuged. Supernatant is withdrawn and its amylose content is determined according to the method of Williams et al. (1970). The value of amylose leaching reflects the association of amylose, and interactions between amylose and amylopectin in the starch. 

The investigations carried out by Professor French and his students were based on sound experimental approaches and on intuitive theoretical considerations. The latter often resulted in new experiments for testing a hypothesis. On the basis of theoretical considerations, Professor French proposed a model for the structure of the amylopectin molecule, and the distribution of the linear chains in this molecule. This model was tested by utilizing enzymes that selectively cleave the linear chains, and the results substantiated the theoretical deductions. He proposed a theory on the nature and types of reactions occurring in the formation of the enzyme - starch complex during the hydrolysis of starch by amylases. In this theory, the idea of multiple attack per single encounter of enzyme with substrate was advanced. The theory has been supported by results from several types of experiments on the hydrolysis of starch with human salivary and porcine pancreatic amylases. The rates of formation of products, and the nature of the products of the action of amylase on starch, were determined at reaction conditions of unfavorable pH, elevated temperatures, and increased viscosity. The nature of the products was found to be dramatically affected by the conditions utilized for the enzymic hydrolysis, and could be accounted for by the theory of the multiple attack per single encounter of substrate and enzyme. 

The molecular weight of amylopectin is appreciably higher, since according to osmotic pressure determinations it is 50,000-1,000,000. 

From the determination of the end groups, the long-chain molecules of amylopectin seem to be composed of 24-30 glucose units, from which it is concluded that the substance is a branched-chain carbohydrate. 

Sen, M., Thevanat, C., and Prioul, J.L. 1997. Simultaneous spectrophotometric determination of amylose and amylopectin in starch from maize kernel by multiwavelength analysis. J. Cereal Sci. 26 211-221. 

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