BN 206: BIOCHEMISTRY PRACTICALS PRACTICAL 2: ISOLATION OF AMYLOSE AND AMYLOPECTIN FROM STARCH

ABSTRACT

            Starch is a carbohydrate consisting of large number of glucose units joined by glycosidic bonds. It is used as energy store in most green plants. It is a most common carbohydrate in human diet and is found in food such maize, potato, wheat and cassava. Starch is made up of two molecules, the helical and linear amylose and the branched Amylopectin. In starch Amylopectin occupy large percentage by mass than amylose.

The purpose of this experiment was to isolate amylose and Amylopectin molecules from sweet potato using basic solution which tends to make Amylopectin to swell. Stress caused by this swelling phenomenon will interrupt the structure organization and allows leaching of amylose molecule to the surrounding solution. The pellets (Amylopectin) will letter be collected by centrifugation and amylose will be precipitated from the solution by saturation with butanol and collected through centrifugation. Finally iodine test was done to test whether the experiment was successful or not.  

INTRODUCTION

Starch is a homopolymer of glucose forming an α-glucosidic chain called a glucosan or glucan. It is the most abundant dietary carbohydrates in cereals, potato, legumes and other vegetables. The two main constituents are Amylose (15%-20%) which has a non-branching helical structure and Amylopectin (80%-85%) which consist a branching chains composed of 24 – 30 glucose residue united by 1,4 linkages in the chains and 1,6 linkages at the branching points (Robert K Murray, Darly K Granner, Peter A Mayes,Victor W Rodwell, 2003)

            Starch is homopolysaccharide, meaning it is comprised by a single type of monomers; in this case the monomer being glucose. Homopolysaccharide serves as a storage form of monosaccharide that is used in as fuels. Starch is important storage polysaccharide in plant cells. They are heavily hydrated. Because they have many exposed hydroxyl groups, available for hydrogen bond with water (David L Nelson, Michael M Cox).  

            Amylose as one of the constituent of starch is a spiral polymer made up of D-glucose units. It is tightly packed structure and this makes it more resistant to digestion than other starch molecules and it is an important form of resistant starch. This property has been exploited by scientist in making effective prebiotics. Prebiotics are non digestible food ingredients that stimulate growth and/or activity of bacteria in a digestive system in a way claimed to be beneficial to health. Amylose is made up of α-1,4 glucose bond molecule. In glucose carbon are numbered starting at aldehyde carbon, so in amylose 1-carbon of glucose molecule is linked to the fourth carbon on the next glucose molecule, hence the name 1,4 glucosidic bond. There are three form amylose chain can take. It can exist in disordered amorphous conformation, it can bind itself in a double helix or it can bind with other hydrophobic guests such as iodine, fatty acid or an aromatic compound. The last form has been utilized and it is the basis of starch test in food substances. Iodine molecule slips inside amylose coil and results in formation of deep blue color. Below is an example of amylose molecule.

            Amylopectin is a highly branched polymer of glucose and it is the one component which makes the soluble part of starch. It is composed of linearly linked alpha 1,4 linked glucose units with occasional alpha 1-6 glycosidic bonds which provide branching  points. Amylopectin is highly branched, formed of 2000 to 200,000 glucose units and its linear chains are formed of 20 to 30 glucose subunits. The significant of branching structure of Amylopectin is that it provide a quick release (or storing) glucose units for (or from) metabolism. Iodine molecule slips inside Amylopectin molecule resulting in formation of red-violet or purplish-red color. In the following page is an example of amylopectin,

            The aim of this experiment was to isolate amylose and amylopectin from sweet potato. This is possible due to the fact that Amylopectin tend to swell under basic condition creating a stress which will result in leaching out amylose.

MATERIAL AND METHODS

Material used

·         Sweet potato

·         Conical flask

·         Water bath

·         Centrifuge machine

Chemical used

·         0.16M NaOH

·         0.6M HCL

·         5% NaCl

·         Iodine

·         1-butanol

EXPERIMENTAL PROCEDURES

1.      Potato was grinded and a 40% (w/v) of aqueous slurry of potato was made using distilled water as solvent

2.      In a flask, 5 mL of 40% (w/v) aqueous slurry  of potato starch followed by 55 mL of 0.16 M NaOH and the mixture was swirled gently until the suspension was clear

3.      After 5 minutes, 15mL of 5% NaCL in a 0.6 M HCL was added and mixed gently.

4.      The mixture was centrifuged at 10,000 rpm for 15 minutes. Then the supernatant was seeped in a separate flask.

5.      The pellets formed were washed by suspending them in a 20 mL 1% NaCL for a week and letter the pellets (Amylopectin) were collected by centrifuging the mixture at 10,000 rpm for 15 minutes.

6.      Amylose from the original supernatant (from 4) was precipitated by saturating it with 1-butanol and let it stand for a week. The amylose was then collected by centrifugation of the mixture at 50,000 rpm for 15 minutes.

RESULTS

IODINE TEST

0.5g of Amylose and amylopectin were dissolved in 2 mL of distilled in two different test tubes. 2-5 drops of iodine solution was added in each test tube and the colour change was observed.  

Starch molecule	Iodine	Color change
Amylose	2 drops	Blue color
Amylopectin	2 drops	Red-violet

 

DISCUSSION

            The reaction of iodine solution with amylose and Amylopectin resulted in color change. In a test tube one containing amylose solution when few drops of iodine was added the solution turned into deep blue color indicating that amylose is present in the solution. Similar to test tube 2 which contains Amylopectin the addition of iodine in it caused the solution to change into red- violet color. This color change is observed due to the fact that iodine tend to fit neatly inside the structure of amylose and Amylopectin. In this experiment other groups have failed to observe color change and this may be due to complication during experimental procedures complications of the instruments such as the centrifuge machine might turn off while time allocated for specific revolutions of the sample is not reached. Improvement of experimental procedure is very important also in getting accurate results for the give experiment. For example, in this experiment the sample were required to stand overnight but instead we left them for a week. If they required to stand overnight, then it important to follow that otherwise the sample must be kept in a refrigerator if they are to stay for some days.

CONCLUSION

            Isolation of Amylose and Amylopectin from starch has helped in understanding the structure and properties of these components in starch. Understanding the structure and properties of starch and its components has lead for it to be used in industrial scale in production of many useful products. For example starch is used in papermaking, used as food additives in food processing industries usually as thickeners and stabilizers and corrugated board adhesives, which all this wouldn’t have been possible without isolation of amylose and Amylopectin from starch so as to study their structure and properties.

QUESTIONS AND ANSWERS

1.      Why sodium hydroxide and 1-bitanol were used in this experiment

Sodium hydroxide is used to make the gel swell before being neutralized while 1-butanol was used to precipitate amylose from the original supernatant.

2.      What is the difference between amylose and Amylopectin?

The difference between amylose and Amylopectin are as follows;

                   I.            Amylose components are insoluble in water while those of Amylopectin are soluble in water.

                II.            Amylose is not connected by any branching and only have alpha 1,4 glycosidic bonds. Amylopectin on the other hand, is connected by branching and also uses alpha 1,4 glycosidic bonds and 1,6 glycosidic linkage at branching points

             III.            With iodine amylose stains deep blue color while with iodine Amylopectin stains purplish red or red violet.

3.      Which of the following carbohydrate is not a polysaccharide; glycogen, amylose, Amylopectin, isomaltose and cellulose.

Among the above carbohydrates Isomaltose is not a polysaccharide. It is a disaccharide similar to maltose with α-1, 6 linkage instead of α-1,4 linkage. It is a reducing sugar. 

REFERENCE AND LITERATURE CITED

David L Nelson, Michael M Cox. Lehninger Principles of Biochemistry, Fourth edition.

Robert K Murray, Darly K Granner, Peter A Mayes,Victor W Rodwell. (2003). Harper's Illustrated Biochemistry. United States of America: McGrawHill Companies.

www.wikipedia.com