Starch Hydrolysis Test (II Method)

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

Introduction
Starch is a polysaccharide found abundantly in plants and 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—amylose 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 a-amylase, which is present in saliva and pancreatic juice, and aids in the digestion of starch in the gastrointestinal tract.

Principle
Starch is a polysaccharide made of 2 components, amylose and amylopectin. Amylose is not truly soluble in water, but forms hydrate micelle, which produce blue when combined with iodine. Amylose produces a characteristic blue color when combined with iodine, but the halide occupies 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 when mixed with iodine.

Materials

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

Procedure
Preparation of starch agar

• 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 200 mL water and heated slowly with constant stirring. Then all of the ingredients are added to it, transferred into a conical flask, and sterilized by autoclaving at 121.5°C for 15 minutes.
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.

Result
Observe your experimental result.