LAB PROJECT 2: ENZYME

BIOCHEMISTRY SBK3013

LAB PROJECT 2: ENZYME

TITLE: THE EFFECT OF AMYLASE ON A STARCHY FOOD

MAUREEN SANTIH ANAK AMBANG	D20141067070
SHARIFAH AWANIS BINTI SYED MOHD ASWAD	D20141067053
HEIDI AMELDA ANAK LAGAT	D20141067086

DATE OF EXPERIMENT: 10 MARCH 2017

LECTURER: ASSOCIATE PROFESSOR ROSMILAH BINTI MISNAN

INSTRUCTOR: NUR ATIEKAH BINTI AZAHARI

Title: The effect of amylase on a starchy food

Objective: Investigating the effect of amylase on a starchy foodstuff.

By working through this practical we learned that:

1.    investigate enzyme action on a foodstuff

2.    see that food must be digested before it can be absorbed in the digestive system

3.    evaluate a model for the process of digestion

Introduction:

Enzymes are protein molecules that are manufactured by all plant and animal cells. All cells require enzymes to survive and function. Enzymes are catalysts, which means that they make chemical reactions go faster, but are not changed by the reaction. For example, digestive enzymes cause food that you eat to be broken down much faster than would occur without them, but they are not broken down in the reaction they are speeding up.

Our teeth break food down into small pieces when we chew. This is only a start to the process of digestion, as chewed pieces of food are still too large to be absorbed by the body. Food has to be broken down chemically into really small particles before it can be absorbed. Enzymes are needed so that this break-down happens quickly enough to be useful.

Enzyme is a special proteins that can break large molecules into small molecules. Different types of enzymes can break down different nutrients which is amylase enzymes that break down starch into sugar, protease enzymes break down proteins into amino acids and lipase enzymes break down fats into fatty acids and glycerol.

Materials:

Starchy foods, boiling tubes, test tubes, visking tubes, elastic bands, dimple tile, thermometer, beaker containing warm water at 37°C, large beaker of boiling water.

(Each group has to bring one starchy food such as rice, bread, cereal etc).

Reagents:

Amylase, Iodine, Benedict reagent.

Procedure:

1. 3 boiling tubes was labelled as 1, 2, 3.

2. 3 model guts is set up:  a wet piece of Visking tubing was taken, a knot was tied in one end.

3. The spatula was used to add the starchy foodstuff to each of the model guts until they are half full.

4. The outside of each piece of Visking tubing was rinsed under a running tap.

5. The starch-filled model gut was filled in a boiling tube.

6. The model gut was placed and secured with an elastic band.

7. The boiling tube was filled with 25 ml warm water.

8. One drop of the warm water we have added Immediately withdrawn  and tested  with iodine on a dimple tile.

9. 5 ml of water was added to model gut 1.

10. 5 ml of amylase was added to model gut 2.

11. 5 ml of treated amylase was added to model gut 3 (depends on group creativity- such as boiled amylase, pH treated amylase etc).

12. All the boiling tubes containing the model guts was placed in a beaker containing water at approximately 37ºC.

13. Incubation time set for 30 minutes.

14. While we were waiting, iodine test and Benedict test wad done, as follows:

·         A grain of starchy food was placed in a well on the white tile and add a drop of iodine. (A blue-black colour indicates that starch is present).

·         Some starchy food was placed in a test tube. 2 ml of water, 2 ml of Benedict’s reagent  added and placed into a large beaker of boiling water. The colour after 2-3 minutes was checked. (An orange (or greeny-yellow) colour indicates that reducing sugar such as glucose or a similar sugar is present).

·         Observations was recorded.

15. After 5 minutes incubation, the iodine and Benedict test was repeated for both water in the gut model and water in the boiling tube, as follows:

·         Some of the water in the model gut 1 was taken and the water surrounding the model gut in boiling tube 1 was taken by using a transfer pipette. 1 drop of these water was dropped in a well of the white tile and a drop of iodine was added. The result was recorded.

·         2 ml of the water from the model gut 1 and 2 ml of the water in the boiling tube 1 was taken. Each water was placed into a test tube. 2 ml of Benedict’s reagent dropped in each test tubes and placed them into the large beaker of boiling water. The colour after 2-3 minutes was recorded.

16. Step 15 was recorded every 5 minutes until 30 minutes.

17. Steps 14 to 16 repeated with water from gut models and boiling tubes 2 and 3. Observation was recorded.

Results:

Before

After

First 5 minutes

Second 5 minutes

Third 5 minutes

Fourth 5 minutes

Fifth 5 minutes

Last 5 minutes

Time taken	Colour inside the visking tube after tested with iodine			Colour outside the visking tube after tested iodine
	Amylase	Treated amylase	Water	Amylase	Treated amylase	Water
5 minutes	Dark blue	Dark blue	Dark blue	Yellow	Yellow	Yellow
10 minutes	Dark blue	Dark blue	Dark blue	Yellow	Yellow	Yellow
15 minutes	Dark blue	Dark blue	Dark blue	Yellow	Yellow	Yellow
20 minutes	Dark blue	Dark blue	Dark blue	Yellow	Yellow	Yellow
25 minutes	Dark blue	Dark blue	Dark blue	Yellow	Yellow	Yellow
30 minutes	Dark blue	Dark blue	Dark blue	Yellow	Yellow	Yellow

Time taken	Colour inside the visking tube after tested with Benedict solution			Colour outside the visking tube after tested Benedict solution
	Amylase	Treated amylase	Water	Amylase	Treated amylase	Water
5 minutes	Red precipitate	Red precipitate	Orange precipitate	Orange precipitate	Orange precipitate	Blue solution
10 minutes	Red precipitate	Red precipitate	Orange precipitate	Orange precipitate	Orange precipitate	Blue solution
15 minutes	Yellow precipitate	Orange precipitate	Orange precipitate	Yellow precipitate	Orange precipitate	Blue solution
20 minutes	Orange precipitate	Brown precipitate	Black precipitate	Orange precipitate	Orange precipitate	Orange precipitate
25 minutes	Orange precipitate	Brown precipitate	Blue solution	Orange precipitate	Orange precipitate	Blue solution
30 minutes	Orange precipitate	Brown precipitate	Blue solution	Orange precipitate	Orange precipitate	Blue solution

Discussion:

Iodine test is used to detect the present of starch in certain substance. Iodine test shows positive results when the colour of iodine change from yellow to dark blue solution. From this experiment, the samples that are inside the visking tube shows a positive results towards iodine test. This means that there are starch present in each sample inside all the visking tube. While, for the water outside the visking tube, all the water samples shows negative results which indicate that there are no starch present in all water samples.

A Benedict’s test is used to determine the presence of reducing sugars such as fructose, glucose, maltose and lactose in certain substance. A positive test with Benedict’s reagent is indicated by a change in colour, often from blue to a brick-red precipitate. In this experiment, when testing for the presence of reducing sugars in food, the food sample is dissolved in water and a minimal amount of food sample is added into Benedict’s solution that we prepared. As a results, only the unchanged blue solution shows that there is no reducing sugar in the water sample that not treated with enzyme. This is because, there is no enzyme to break down starch into sugar. As for the sample that treated with enzyme amylase, a positive result appeared but with different colour. Different colour of solution indicate that different amount of reducing sugar in each samples.

Questions:

1 What does the Visking tubing of the model gut represent?

Visking tube act as the wall of small intestine in our stomach.

2 What does the water in the boiling tube (outside the model gut), represent?

The water represents the blood in the capillaries outside the intestine.

3 How is this model different from the situation in a real gut?

The differences between Visking tubing and a real gut are the Visking tubing membrane is smooth, a gut lining is folded and folded again with a larger surface area for its length.

4 What has happened to the starch in each of your tubes?

Starch in the water medium remain unchanged while starch in amylase and treated amylase is broke down into sugar.

5 What has happened to sugars (detected with Benedict’s reagent) in each of your tubes?

The sugar shows positive results when tested with Benedict’s reagent.

6 Why does the body need enzymes to digest food?

We need enzyme to digest our food so it can be absorbed through the walls of the small intestine into the blood stream.

7 What is your result with treated amylase? Explain.

In treated amylase samples, the amount of reducing sugar is high compare to other test tube. This is because, treated amylase break down the starch into sugar a lot more faster that amylase.

Conclusion:
From this experiment, we can conclude that amylase is an important enzyme that responsible in breaking down carbohydrates into smaller molecule so that the smaller molecules can be abserbed into our body. without amylase, our body cannot break down carbohydrates as a food sources into our body.

References: 
1. Peter Layer, Alan R. Zinsmeister, Eugene P. DiMagno; Effects of decreasing intraluminal amylase activity on starch digestion and postprandial gastrointestinal function in humans; Volume 91, Issue 1; July 1986; Elsevier.
2. Peter J. Butterworth, Frederick J. Warren, Peter R. Ellis; Human α-amylase and starch digestion: An interesting marriage; Volume 63, Issue 7; July 2011; Wiley Online Library.

Reflection:
Maureen Santih:

The experiment is quite interesting because I love the colour changed when we put the solution from inside the visking tube and out of visking tube into the water bath. In this experiment,we used plain cracker which is quite easy to put inside the visking tube after we put some water on it. This experiment also required patience because we need to take the sample from inside and out of the visking tube to be tested for every 5 minutes until 30 minutes. From this experiment, I get to know that the Benedict’s solution will turn from blue red,orange and green if there is presence of sugar. The iodine will change from yellowish brown to dark blue if there is any starch presence.

heidi amelda:

From this experiment, we brought our own starchy food (crackers) to examine the present of starch. I have learnt how the amylase affect the starchy food. This experiment took a long time because we have to repeat the step every 5 minutes within 30 minutes. During this experiment, we have done few mistake such as we put the wrong solution and wrong starchy food to the solution in the test tube. we have to do it again. It really need a lot time and patience.

Sharifah Awanis:

In this experiment, we used cracker biscuits as starchy food to be examined. this experiment was interesting as i can see a lot of colour changes. The colour changed also can be differ when we put the test tube with Benedict's reagent into the hot water bath. So, i begin to do some reading to find out what actually caused that to happened as i was curious abouit it. Although this experiment takes a long time as it going through a lot of preparation because we need to repeatedly prepared the Benedict's reagent solution, we enjoy this experiment because we got all the results.