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Digestion
*Hydrolysis Reactions*
(Lab 6)
Ingested food contains macromolecules such as proteins, polysaccharides, and fats (triglycerides)
which are too large to be absorbed into the bloodstream from the lumen of the small intestine. These
molecules must be broken down by chemical digestion into smaller molecules which can be absorbed.
Chemical digestion involves hydrolytic reactions mediated by appropriate enzymes. The end-products
of digestion are monosaccharides from polysaccharides, amino acids from proteins, and glycerol and
fatty acids from fats. Once absorbed into the blood, these molecules are transported throughout the
body and enter cells by the processes of diffusion, facilitated diffusion, and/or active transport. Cells use
these molecules to synthesize new molecules (anabolism) or divert them to the glycolysis/respiratory
pathways to be broken down (catabolism) in order to liberate stored energy for the production of ATP.
The numerous chemical reactions involved in anabolism and catabolism collectively make up an
organism’s metabolism.
Laboratory Objectives:
1. Identify the enzyme used in lab to digest starch.
2. Identify the end-products of starch digestion.
3. Describe the procedure for the Benedict’s test for reducing sugars and name the reagent
used; describe the appearance of a positive and negative test and interpret given results.
4. Describe the test for starch, naming the reagent used, and describing a positive and
negative test; interpret given results.
5. Explain the exercise done in lab that demonstrated starch digestion.
6. Identify the enzyme used in lab to digest the protein albumin; describe the conditions
needed for activity of this enzyme; explain the result of the exercises done lab.
7. Identify the end-products of protein digestion
8. Describe the exercise done in lab to demonstrate lipid digestion; explain the appearance
of the results.
9. Identify the end-products of lipid digestion
10. Define: hydrolysis, protease, and lipase
EXERCISE:
Materials for Part A:
disposable test tubes for saliva collection
2% α-amylase solution (alternative to saliva)
2 test tubes for starch and sugar tests
test tube rack
graduated cylinder
150-mL beaker
600-mL beaker
1 5-inch piece of dialysis tubing
2 pieces of string
starch solution
iodine solution
Benedict’s solution
thermometer
hot plate
10-mL pipet
pipet pump
400-mL beaker to be used as a boiling
water bath
scissors
parafilm
lab coat
gloves
disinfectant
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Materials for Part B:
Hard-boiled egg white
10% hydrochloric acid (HCl)
3% pepsin solution
five test tubes
five rubber stoppers
test tube rack
tape and pens for labeling
hot plate
10-mL pipet
400-mL beaker
test tube holder
scalpel
pH paper
Materials for Part C:
6 petri plates of Tributyrin Agar (1 for each lab table)
inoculating loop
incinerator
living culture of a lipolytic bacterium (a bacterium that produces lipases)
living culture of a non-lipolytic bacterium (a bacterium that does not produce lipases)
Part A: Carbohydrate Digestion
Each student must wear a lab coat and gloves.
Only soluble materials diffuse through membranes. Starch is insoluble but can be converted to
soluble sugars such as maltose and glucose by the action of salivary amylase, an enzyme
present in saliva.
Procedure:
1. Set up a boiling water bath for this experiment. Fill a 400-mL beaker ~2/3 full with water
and heat it on a hot plate. Turn the hot plate dial to the highest heat setting.
2. Collect 5 mL of saliva (per group). If desired, chew a piece of parafilm or chewing gum to
increase production of saliva. Each student must use their own disposable test tube for the
initial saliva collection. Pour all saliva into a single graduated cylinder for volume
measurement. Save the saliva for Step #5 below. (Alternative: Instead of saliva, transfer ~3
mL pure amylase enzyme directly into the bag  Step #5 below.)
3. Moisten the dialysis tubing under running tap water to rehydrate it and securely tie one end
of the tubing with a piece of string. The tie should be about 1 cm from the end of the tubing.
4. Add 5 mL of starch preparation and 10 drops (approximately 0.5 mL) of iodine solution to
the dialysis tube. Note the appearance of the contents of the dialysis tube (here and in
Table 6.2 in the Results Section):
________________________________________________________________________
5. Add the 5 mL of saliva (OR ~3mL of 2% -amylase solution) to the dialysis tube.
6. Securely tie the open end of the dialysis tube, eliminating as much air as possible, and rinse
the exterior of the bag.
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7. Place the dialysis tube into a 150-mL beaker; add just enough 37C to 40C tap water to
cover the tube.
8. Make a 40C water bath by adding ~300 mL of 40C tap water to a 600-mL beaker. Float
the 150-mL beaker containing the dialysis bag in the 600-mL beaker. Maintain the
temperature of the water in the 600-mL beaker at 37C to 40C by adding hot water from the
boiling water bath (set up in Step #1) as necessary. Use a 10-mL pipet fitted with a pipet
pump to transfer hot water to the water bath. Note: DO NOT allow the temperature of the
water bath to exceed 40C, and DO NOT transfer boiling water directly to the 150-mL
beaker.
9. After 45 minutes, remove the 150-mL beaker from the 600-mL water bath. Set it on the
bench and test the water outside of the bag (in the 150-mL beaker) for sugar and starch:
For the starch test: Remove 10 mL of sample to a test tube and add 5 drops of Lugol’s
iodine solution. Prepare a control tube using 10 mL of tap water and 5 drops of iodine.
Formation of a blue-black color is a positive test for starch. Compare results in your
experimental tube versus the control tube.
For the sugar test (The Benedict's Test): Remove 10 mL of sample to a clean test tube and
add 10 drops of Benedict’s solution. Prepare a control tube by adding 10 mL of tap water
to a clean test tube with 10 drops of Benedict’s solution. Heat both tubes in a boiling water
bath for ~5 minutes. A change in color from clear blue to cloudy green, yellow, orange, or
red is a positive test for sugar. Refer to Table 6.1 below to aid in your interpretation of the
Benedict’s Test.
Table 6.1 Benedict’s Test Results
Observation
Interpretation
solution remains blue
solution turns green
solution turns yellow
solution turns orange
solution turns red
- (negative test result)
+ (slightly positive)
++ (positive)
+++ (strong positive)
++++ (very strong positive)
10. Record your results in the results section below.
NOTE: Discard the contents of the test tubes in the waste containers as directed by
the instructor. Use a test tube holder to handle the test tubes in the boiling water
bath. Turn off the hot plate and leave it on the table to cool. Allow the beaker of
boiling water to cool completely before discarding the water. If SALIVA was used 
The dialysis bag (and any other disposable items contaminated with saliva) should be
discarded in a biohazard bag. The glassware (150-mL beaker and graduated cylinder
used to measure the volume of saliva) should be bleached or autoclaved before
washing.
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RESULTS:
1. Draw a diagram (Figure 6.1 below) to illustrate what happened in this exercise. Compare
the appearance of the contents of the dialysis bag at the beginning and at the end of the
exercise. Explain any changes that may have occurred. Illustrate the molecular changes
that occurred inside the dialysis bag.
Figure 6.1
Beginning of experiment:
Conclusion of experiment:
Table 6.2
Solution
Source
Initial
Color
Final
Color
Starch Test
(note color and +/- reaction)
Benedicts (Glucose) Test
(note color and +/reaction)
Dialysis
Bag
Beaker
Solution
Water
Control
2. Describe why each solution in the table above was the color observed during the experiment
and after the starch and Benedict’s test.
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Part B: Protein Digestion
Enzymes are most often proteins. Enzymes that hydrolyze other proteins into amino acids are
called proteases. Pepsin is a protease that is activated in an acid medium. Egg white is
composed of a protein called albumin. In this lab, we will examine proteolytic digestion of
albumin by pepsin.
The contents of the five tubes is summarized in the following table:
Table 6.3 Experimental Design
Tube A
Tube B
Tube C
Tube D
Tube E
10 mL
10 mL
10 mL
10 mL
10 mL
10% HCl
--
5 drops
--
5 drops
5 drops
Pepsin
--
--
25 drops
25 drops
25 drops
Water
pH
Procedure:
1. Using tape and a pen, label the five test tubes A, B, C, D, and E. Use Table 6.3 as a guide
as you add the appropriate components to each tube.
2. Add 10 mL of water to each tube.
3. LAB COAT & GLOVES required:
Tubes B, D, and E: add 5 drops of 10% Hydrochloric acid (HCl) to each.
4. Tubes C, D, and E: add 25 drops (or 2 mL) of pepsin solution to each.
5. Use the boiling water bath set up in the previous experiment (Part A; 400-mL beaker).
Place Tube E into the boiling water bath until the contents of the tube become cloudy (~5
minutes). Using a test tube holder, remove the tube from the water bath and set it in the test
tube rack to cool to room temperature.
6. Tap the tubes gently to mix the solutions. Test the pH of each tube (pH paper) and record
results in Table 6.3 above and in Table 6.4 of the Results section. If necessary, add extra
HCl to tubes B, D, and E (you want the pH of these tubes to be in the approximate range of
2.5 to 3.5).
7. Using a scalpel, cut 5 cubes of egg white, no larger than 3 mm on each side. Place one
cube of egg white into each of the five tubes.
8. Close each tube with a rubber stopper, label your rack of tubes by group name, and
incubate the rack of tubes in the lab cabinets until the next lab period.
**STOP HERE for Part B**
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Part B continued:
9. Carefully observe the contents of each tube, noting any changes in appearance. Using pH
paper, test the pH of each tube. Record your pH results and observations in Table 6.4 in
the Results section.
RESULTS:
Table 6.4
Tube
Initial
pH
Final
pH
Final Observations
(Appearance of Tube Contents)
Hydrolysis of Protein?
(Yes/No)
A
B
C
D
E
1. Summarize your results:
2. Describe the controls in this experiment. Explain the purpose they serve.
3. What is the purpose of the pepsin?
4. What is the purpose of the hydrochloric acid (HCl)?
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Part C: Lipid Digestion
Tributyrin is a fat which is opaque when incorporated into an agar growth medium. Enzymes
which hydrolyze fats are called lipases. The end-products of fat digestion are glycerol and fatty
acids. Glycerol and fatty acids do not appear opaque when they are present in an agar growth
medium.
Procedure:
1. The instructor will inoculate the Tributyrin Agar plates. Each plate will be inoculated on one
half with the lipolytic bacterium and on the other half with the non-lipolytic bacterium.
2. Incubate the plates at the appropriate growth temperature for the bacteria (37ºC) until the
next lab period.
RESULTS:
1. Observe the two plates and describe the appearance of both. (Explain the appearance of
the medium surrounding the bacteria.)
2. Did digestion of the lipid occur inside the bacterial cells or outside the bacterial cells in the
growth medium? Since you cannot see individual cells without a microscope, how do you
know where the digestion took place?