Treatment Of Starch With Acid Biology Essay

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Starch is a polymer, a group of polysaccharides, composed of glucose molecules that liked with a(1,4) glycosidic bonds and oxygen bridges. 80% of starch is amylopectin which is a water-soluble compound.

Starch can be digested by mammalians, unlike cellulose. Their difference comes from the enzyme-substrate relation. Linkage between glucose molecules which cellulose contain b(1,6) glycosidic bond cannot be recognized by the digestion enzyme(amylase) but starch can which contain a(1,4) glycosidic bond.

Treatment of starch with acid by addition of some enzymes causes hydrolization of starch and yields dextrin, continued hydrolization produce dextrin and finally D-glucose.

Iodine is a starch indicator. When combines with starch blue black color occur and this can be detected and measured by using a spectrometer.

Fig.1: Basic structure of a starch chain [1]

Amylase enzyme

Amylase is extreted by pancreas and salivary glands can digest some carbohydrates by hydrolyzing amylopectin and amylase. There are several factors that affect enzyme activity like pH, temperature, substrate or enzyme concentration, and substrate.

Spectrophotometry

Spectrometer measures the transmission or absorption of liquids or solids as a function of wavelength; determines the absorption spectrum of a pure substance in a solution, and detects the concentration of the substance.

I: incident light

I0=transmitted light

T= I/I0 = Transmittance

%T= I/I0 *100

ABS=log(100/%T)

APPARATUS

Equipments Used in The Experiment

Test tubes

Test tube rack

Pasteur pipettes

Pipettes

Beaker

Plastic cuvettes

Chemicals Used in The Experiment

Human salivary enzyme

Starch solution 20g/L

HCl Stopping solution, 0,1N HCl

Iodine Reagent Stock Solution(in aqueous solution)(dilutet to 1:100)

Iodine(I2): 5g/L

KI:50g/L

Potassium phosphate buffers

KH2PO4 (Monobasic Phosphate) (MW=136.1)

K2HPO4·3H2O (Dibasic Phosphate) (MW=228.23)

PROCEDURE

Preparation of Enzyme, Starch and Water baths

1mL of human salive was diluted with 9mL of water.

20g of soluble potato starch was mixed in 50mL of cold water.

The starch mixture was added in 900mL of boiling water and stirred gently.

Gelatinized solution was mixed well and cooled to room temperature. Water amount in the starch mixture completed to 1L.

1mL of human saliva was diluted with 9mL of water.

4 water baths were prepared, water temperatures were 30°, 50°, 70° and 90°.

PH Effect

Solutions with different pH were prepared by adding 20mL of 0.1M KH2PO4 to make pH5, 20mL for pH6, 100mL for pH7; 20mL of Na2HPO4 for pH 8 and 20mL for pH9. Necessary amount of HCl solution was added to reach desired pH values while the solutions were measuring with a pH meter.

5mL of starch solution was added in each tube that contains 5mL of buffer (different pH for each tube).

1mL of saliva dilute was added in each tube to start digestion reaction. Exactly 10 minutes later 0.5mL of each sample was taken to another tube and added 5mL HCl solution to stop the reaction (0.1N).

0.5mL of mixtures was transferred to another tube that contains 5mL of iodine solution.

Absorbance values of solutions were measured at 620nm by using spectrophotometer.

Temperature Effect

5mL of pH7 buffer was added in 4 different tubes and the tubes were marked.

5mL of starch solution 20g/L was added in each tube.

1mL of salivary dilute was added in each tube, the tubes were placed in the water baths that each has different temperature degrees.(prepared at i.6th step)

The reactions were stopped exactly 10 minutes later.

Absorbance values were measured, and their color changes were noted.

RESULTS

Temperature (°C)

Absorbance (at 620nm)

30

0,030

50

0,015

70

0,048

90

0,167

Table 1. Temperature & Absorbance

Fig. 2: The graphic of absorbance change with temperature

pH

Absorbance

(at 620nm)

5

0,104

6

0,046

7

0,042

8

0,049

9

0,087

Table 2. pH & Absorbance

Fig. 3: Absorbance change depend on pH value

DISCUSSION

In this experiment enzyme activity of amylase on starch at different conditions are determined.

COOH and NH2 groups on R group of starch molecule are deformed by the enzyme and tertiary structure broken, and denaturation decreases resolution, absorbance of fluorescent light is affected. In aqueous medium hydrophobic parts of protein escape from water and hydrophilic parts folded outside. Denaturation of the molecular structure cause the hydrophobic parts go outside and the proteins that affect each other aggregates by making clusters.

The spectrometric analyze detects the enzyme activity by measuring increasing product and decreasing substrate amount. Additionally in this experiment optimum pH and temperature conditions are observed respect to enzyme activity by keeping starch amount constant bur changing buffer concentrations and temperatures.

According to the results between different pH values (5,6,7,8,9), the range between pH6-7 gives the best results( see fig.2) which is the pH range of a human mouth. On the other hand the enzyme shows better results between 30°C to 50°C temperature values (see fig. 3) which includes the body temperature range of a human.

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