Digestive Enzymes Lab
Purpose: to visualize the products of enzyme digestion in the human gastrointestinal tract. Activity 1 highlights the
digestion of protein in the stomach by the enzyme pepsin. Activity 2 illustrates the digestion of milk fat by intestinal
lipase into fatty acids and the resulting change in pH. Activity 3 uses iodine and Benedict’s reagent to visualize the
digestion of starch to glucose by amylase in the mouth.
Pre-lab Questions:
1. Define the term enzyme.
2. For each of the following enzymes, describe the type of food digested, the essential nutrient that results from the
digestion, and the organ where digestion occurs: A. amylase B. pepsin
C. lipase
Copy the following data table into your lab notebook:
Test Contents
tube
1
Protein and biuret (positive control)
2
Protein, pepsin, and biuret (at pH 1.5)
3
Protein, pepsin, and biuret (at pH 12)
4
Litmus-milk solution
5
Litmus-milk and lipase solution
6
Starch and iodine (positive control)
7
Starch, amylase, and iodine
8
Starch, amylase, and Benedict’s
9
Glucose and Benedict’s (positive control)
10
Starch and Benedict’s (negative control)
Observations
Activity 1 –Protein Digestion
1. Use a wax pencil to label 3 test tubes 1, 2 and 3.
2. Use a clean, graduated pipet to add 3 mL of the 2% protein solution to test tube 1.
3. Add 1 mL of the 2% protein solution to test tubes 2 and 3.
4. Use a clean, graduated pipet to add 2 mL of the 1% pepsin solution to test tubes 2 and 3. Gently swirl the test tube to
mix the contents.
5. Use a clean, graduated pipet to add 1 mL of 0.1 M NaOH to test tube 3.
6. Place all 3 test tubes in a 40⁰C water bath for 15 minutes
7. Remove the test tubes from the water bath and use a clean, graduated pipet to add 1 mL of biuret test solution to
each test tube.
8. Observe the color changes and appearance of the resulting solution in each test tube. Record the observations in a
data table in your lab notebook. NOTE: Biuret test solution is bluish-purple in the presence of polypeptides and a
purplish-pink color in the presence of amino acids.
Activity 2 – Fat Digestion
9. Use a wax pencil to label 2 test tubes 4 and 5.
10. Use a clean, graduated pipet to add 2 mL of the 1% litmus-milk solution to test tube 4. This solution contains
buttermilk, a fat.
11. Add 1 mL of the litmus-milk solution to test tube 5.
12. Use a clean, graduated pipet to add 1 mL of the 1% lipase solution to test tube 5 only. Gently swirl to mix the
contents.
13. After 3 minutes, record the color of the solutions in your data table. NOTE: Litmus is a pH indicator. Litmus appears
blue in basics solutions and pink in acidic solutions.
Activity 3 – Carbohydrate Digestion
14. Use a wax pencil to label 5 test tubes 6-10.
15. Use a clean, graduated pipet to add 1 mL of the 1% starch solution to test tubes 6, 7, 8, and 10 only.
16. Use a clean, graduated pipet to add 1 mL of the 1% amylase solution to test tubes 7 and 8 only.
17. Use a clean, graduated pipet to add 1 mL of the 1% glucose solution to test tube 9 only.
18. Gently swirl the test tubes to mix the contents.
19. Allow the test tubes to sit undisturbed for 2 minutes.
20. Test the starch-amylase solution for presence of starch:
a. Use a clean, graduated pipet to add 4-6 drops of iodine solution to test tubes 6 and 7.
b. Record the color of the resulting solutions in your data table. NOTE: Test tube 6 is your positive control and
the blue-black color indicates the presence of starch.
21. Test the starch-amylase solution for glucose:
a. Use a clean, graduated pipet to add 1 mL of Benedict’s reagent to test tubes 8, 9, and 10.
b. Place the test tubes in a boiling water bath using a test tube clamp.
c. After 2-3 minutes, remove the test tubes from the hot water bath using a test tube clamp.
d. Record the color of the resulting solutions in your data table. NOTE: Benedict’s reagent contains Cu2+ (aq)
which, when heated, reacts with glucose to form a red/orange/or yellow colored precipitate. Benedict’s will not react
with disaccharides or polysaccharides.
Post-Lab Questions. Put the answers to the following in your lab notebook in complete sentences:
1. Compare and contrast the results of the biuret test results for test tubes 1 and 2. Describe the evidence, if any, for
the digestion of protein using pepsin.
2. The pepsin solution in test tube 2 was prepared using 0.01M HCl. Explain the difference in your results from test
tubes 2 and 3. Relate this to the location of pepsin secretion in the human body.
3. Compare and contrast the observations of the test results for test tubes 4 and 5. Describe the evidence, if any, for the
digestion of butterfat using lipase.
4. Compare and contrast the iodine test results for starch (tube 6) and the starch/amylase (tube 7). Explain the test
results based on the activity of amylase.
5. Explain the results of the Benedict’s test for tubes 8-10. What are the products of the digestion of starch by amylase?
6. Summarize the digestion of a pepperoni pizza served with a glass of milk. Indicate the enzymes responsible for
digestion in the mouth, stomach, and small intestine.