Starch Hydrolysis Test

Aim
To study the hydrolysis of starch by microorganisms by the production of the enzyme amylase.

Introduction
Starch is a polysaccharide found abundantly in plants, and is usually deposited in the form of large granules in the cytoplasm of the cell. Starch granules can be isolated from the cell extracts by differential centrifugation. Starch consists of 2 components—amylase and amylopectin, which are present in various amounts. The amylase consists of D-glucose units linked in a linear fashion by α-1,4 linkages. It has 2 nonreducing ends and a reducing end. Amylopectin is a branched polysaccharide. In these molecules, shorter chains of glucose units linked by α-1,4 are also joined to each other by α-1,6 linkages. The major component of starch can be hydrolyzed by α-amylase, which is present in saliva and pancreatics juice and participates in digestion of starch in the gastrointestinal tract.

Principle
Starch is a polysaccharide made of 2 components, namely amylase and amylopectin. Amylose is not truly soluble in water, but forms hydrated micelle, which produces blue color with iodine. Amylose produces a characteristic blue color with iodine, but the halide occupy a position in the interior of a helical coil of glucose units. This happens when amylase is suspended in water. Amylopectin yields a micellar which produces a violet color with iodine.

Materials

• Petri plates
• Conical flasks
• Starch agar media
• Bacterial specimen
• Iodine

Procedure
Preparation of starch agar Composition

• Beef extract - 3 g
• Agar agar - 15 g
• Starch - 3 g
• Tryptone - 5 g
• Distilled water - 1000 mL
• PH - 7

Steps

1. Soluble starch is dissolved in a small amount of water and is heated slowly with constant stirring. Then all the ingredients are added to it and transferred into a conical flask and sterilized by autoclaving at 121.5°C for 15 min.
2. The sterilized agar medium is poured into the sterilized Petri plates and allowed to solidify.
3. Each plate is inoculated at the center with the bacterial inoculum.
4. Plates are incubated at 37°C for 24–48 hrs.
5. To test the hydrolysis of starch, each plate is flooded with iodine.