In the lab, the ratio of amylose to amylopectin will be assessed. Some steps of
ID: 218234 • Letter: I
Question
In the lab, the ratio of amylose to amylopectin will be assessed. Some steps of the assay are listed below. Explain what happens in each step!
Starch is extracted with 95% ethanol. The ethanol is then discarded. This step is more important when analyzing flours, but should be performed for starches too. What is accomplished in this step (Hint: think of other components that could be present in the samples)?
Addition of concanavalin A
Treatment with
a-amylase
Amyloglucosidase
Glucose oxidase
Peroxidase
Why are the samples heated before addition of a-amylase and amyloglucosidase?
How would a raw starch differ from a gelatinized starch in terms of digestibility?
Explanation / Answer
Amylose is known as a linear polymer, but is not defined as just a straight chain molecule. It frequently forms a helix and is thought to intertwine even through the several layers of amylopectin. Not only has the amylose this unique shaping, but it has been shown that it consists also of limited but distinctly measurable branch points. Amylose consists predominately of ?-l,4-D-glucose bonds. Amylose also forms a very strong complex with iodine. This complex yields a very intense blue color producing a ?max = 620 nm. Because of this characteristic one can use a light microscope to assay qualitatively for amylose in starch.
Amylopectin (CAS# 9037-22-3) is a highly branched polymer of glucose found in plants. It is one of the two components of starch, the other being amylose. It is insoluble in water. Its counterpart in animals is glycogen which has the same composition and structure, but with more extensive branching that occurs every 8 to 12 glucose units. Starch is made of about 80% amylopectin. Amylopectin is highly branched, being formed of 2, 000 to 200, 000 glucose units. Its inner chains are formed of 20-24 glucose subunits. The glucose residues are linked through alpha-1, 4 glycosidic linkages.
The overall principle is:
Starch samples are completely dispersed by heating in dimethyl sulphoxide (DMSO). Lipids are removed by precipitating the starch in ethanol and recovering the precipitated starch. After dissolution of the precipitated sample in an acetate/salt solution, amylopectin is specifically precipitated by the addition of Con A and removed by centrifugation. The amylose, in an aliquot of the supernatant, is enzymically hydrolysed to D-glucose, which is analysed using glucose oxidase/peroxidase reagent. The total starch, in a separate aliquot of the acetate/salt solution, is similarly hydrolysed to D-glucose and 1 measured colourimetrically by glucose oxidase/peroxidase. The concentration of amylose in the starch sample is estimated as the ratio of GOPOD absorbance at 510 nm of the supernatant of the Con A precipitated sample to that of the total starch sample.
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