LAB EXERCISE 7
DIGESTION
(Seeley 36, 43-49, 872-876, 881-884, 890, 896-901)
Work in groups of 4
Objectives:
 Identify the types of carbohydrates, fats and proteins present in the diet, state their general formulas,
and outline the reactions by which they are digested to absorbable end-products.
 Identify the enzymes of the GI tract which act on each group of food substances, state their origin,
activators (if present), substrates and end-products.
 State the pH in each part of the GI tract where digestion occurs, outline the mechanisms whereby it is
established, and state its significance.
 Explain the role of bile salts in digestion, and state their origin.
SAFETY (for Exercise C):



Do not burn yourself with boiling water or the reagent when you perform the Benedict test.
Use the test tube clamps to pick up test tubes from the beakers on the hot plates.
Put the test tubes inside a test tube holder before carrying them to your bench.
I. DIGESTION OF FATS
A. THE DIGESTION OF MILK FATS BY PANCREATIC LIPASE
EXERCISE A
Equipment:
 bile salts (powder)
 pancreatin solution (0.5% - contains pancreatic lipase)
 cream
 litmus solution (1%)
 distilled water
 pipettes
 pipette suction pump
 test tubes
 water bath at 37 oC
1. Label four test tubes #1A - 2A - 3A - 4A, and add solutions as designated in Table 1. Add the enzyme last!
Table 1. Digestion Tests with Lipase and Bile Salts
Test
Tube #
SUBSTRATE
(Cream)
BILE
SALTS
DISTILLED
WATER
ENZYME
(Pancreatin Solution)
Add last
INDICATOR
(Litmus Solution)
1A
3.0 ml
pinch
-
3.0 ml
3.0 ml
2A
3.0 ml
pinch
3.0 ml
3.0 ml
-
3A
3.0 ml
-
-
3.0 ml
3.0 ml
4A
-
pinch
3.0 ml
3.0 ml
3.0 ml
1
Notes:
i) Litmus is a pH indicator which changes colour from red (below pH 4.5) through various shades of violet to blue
(above pH 8.3). Try it yourself: take a test tube, put approximately 1 ml of litmus solution. The color in the test
tube is blue. Add a few drops of 2N HCl. Is there a change of colour? If yes what is the colour?
.
ii) Enzymatic digestion of fat (triglyceride) releases fatty acids (e.g. butyric acid) which react with litmus and change
its color to red.
iii) Pancreatin is a commercial preparation of dried pancreatic enzymes.
2. Shake the tubes to mix thoroughly, make note of the colour of each tube, and place in a water bath at 37 oC.
Incubate for 1 to 1 1/2 hours.
3. Shake tubes and record their colour in Table 2 every 5 minutes for the first 15, then for every 15 minutes until
no colour change is observed between colour tests.
Choose terms to describe the colours such as: milky blue, milky violet, milky purple, pinkish purple, reddish
purple, transparent blue, transparent red… Write your results in Table 2.
Table 2. Digestion of milk fat by pancreatin: changes in colour.
Colors in test tubes during incubation
Test
tube #
0min
5min
10min
15min
30min
45min
1hour
1 hr 15
min
1A
2A
3A
4A
4. Complete the table below:
Test
tube #
Presence of
fatty acids
(yes/no)
Is the digestion of cream,
fast, slow or is there no
digestion at all?
Brief explanation of each result
1A
2A
2
1.5 hr
3A
4A
3
B. DEMONSTRATION OF THE EMULSIFYING PROPERTIES OF BILE SALTS
EXERCISE B
Equipment:
 bile salts (powder)
 distilled water
 oil
1. Prepare two test tubes:
In the first one, put 3.0 ml. olive oil and 3.0 ml. distilled water.
In the second test tube, put 3.0 ml water and add a pinch of bile salts. Shake vigorously. Then add 3.0 ml of oil.
2. When both test tubes are ready, take one in each hand and shake them at the same time. Put them back in your
tray and observe any changes over time. Note generally how long it takes for the oil and water to separate.
Keep both test tubes until the end of the laboratory period. Do NOT shake them again. Watch for and note any
changes that occur during this time.
II. DIGESTION OF CARBOHYDRATES
EXERCISE C
Equipment:
 starch solution (1%)
 distilled water
 2N HCl
 pancreatin solution (3% - contains pancreatic amylase)
 test tubes
 paraffin
 pH paper
 water bath at 37oC
 iodine solution
 glucose solution (1%)
 maltose solution (1%)
 Benedict's solution
 spot plate
 beaker (Pyrex, for boiling water)
 hot plate
 test tube clamp
THE DIGESTION OF STARCH BY AMYLASE
1. Label four test tubes #1C - 2C - 3C - 4C and add solutions as described in Table 3. Add the enzyme last.
Table 3. Digestion Tests with Amylase
Test
Tube #
OTHER REAGENTS
SUBSTRATE
(starch solution)
ENZYME (Pancreatin)
1C
5.O ml
---
5.0 ml
2C
5.0 ml
5.0 ml dist. Water
---
4
3C
---
5.0 ml dist. Water
5.0 ml
4C
5.0 ml
5 drops 2N HCl
5.0 ml
2. Shake the tubes and determine and record the pH of each reaction mixture in table 4.
3. Place the test tubes in a water bath at 37oC and incubate for at least 45 minutes. Shake the tubes every 15
minutes. (Complete the iodine test and Benedict’s test control test tubes as instructed below while waiting for
these tubes to incubate for the 45 minutes)
4. Divide each of the 4 samples by pouring half of it into a clean test tube (label).
5. Test one set of 4 test tubes for starch by adding 5 drops of iodine solution. Shake and record the color of the
iodine solution in table 4. See the control tests below to analyze results of iodine test.
6. Test the second set of 4 test tubes for reducing sugar by adding 5 ml. of Benedict's reagent to each, and placing
in a boiling water bath for 5 min. Remove and rate the precipitate for colour according to the following scale:
Blue Green +
Yellow ++
Orange +++
Red ++++
Record the color of the precipitate in Table 4. See the control tests below to analyze results of Benedict’s test.
Iodine solution indicates the presence of starch by turning blue. Try it yourself!
- Prepare three test tubes. Place 2ml of 1% starch solution in one test tube, 2ml of 1% glucose solution in a
second, and 2ml of 1% maltose in the third.
- Add 5 drops of iodine solution to each test tube and shake.
Iodine solution is yellow. Is there any change in color after it is added to the glucose solution? If yes, what color is
formed?
Is there any change in color after it is added to the glucose solution? If yes, what color is formed?
Is there any change in color after it is added to the maltose solution? If yes, what color is formed?
Keep these test tubes as controls to compare to the test tubes prepared for starch digestion.
Benedict’s reagent tests for the presence of glucose and other reducing sugars. Reducing
sugars (glucose, fructose, maltose, lactose, etc.) react with Cu+2 ions (blue, soluble) in
Benedict's reagent to form Cu+, which combines with O2 to form Cu2O (red, insoluble).
Non-reducing sugars (starch, galactose, sucrose) do not react with Cu+2. Try it yourself!
- Prepare three test tubes by adding 5 ml of Benedict's reagent to each test tube
- Place 4ml of 1% starch solution in one test tube, 4ml of 1% glucose solution in a second, and 4ml of 1%
maltose in the third.
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- Heat in a boiling water bath for 5 min.
- Cool.
A green, yellow or red precipitate should form if glucose is present. The extent of the colour change is related to the
amount of red Cu+ formed in relation to the amount of blue Cu++ remaining. This is determined by the amount of
glucose present.
Record colour of the precipitate for starch:
Record colour of the precipitate for glucose:
Record colour of the precipitate for maltose:
Keep these test tubes as controls to compare to the test tubes prepared for starch digestion.
Table 4. Digestion of starch by amylase.
Test
tube
#
pH
Iodine
test
Benedict
test
Presence
of starch
(yes/no)
Presence of
reducing sugar
(yes/no)
1C
2C
3C
4C
III. DIGESTION OF PROTEINS
EXERCISE D
Equipment:
6
Brief explanation of each result

Capillary tubes which have been completely filled with coagulated egg white and incubated at 37 oC for 24 hours
in the following solutions:
(1) pepsin in buffer, pH 2.0
(2) pepsin in buffer, pH 4.0
(3) pepsin in buffer, pH 6.0
(4) pepsin in buffer, pH 8.0
(5) buffer, pH 2.0
(6) buffer, pH 4.0
(7) buffer, pH 6.0
(8) buffer, pH 8.0
(9) trypsin in buffer, pH 2.0
(10) trypsin in buffer, pH 4.0
(11) trypsin in buffer, pH 6.0
(12) trypsin in buffer, pH 8.0
Ruler

1. Obtain results from the teaching staff.
2. Each of these tubes was totally filled with coagulated egg white. Determine the amount of egg white that has
disappeared by measuring the length of empty tube at each end in mm, summing them and expressing them in
percentage of the total length of the capillary tube (tubes were completely filled with egg white at the start of the
experiment).
3. Tabulate results. Compare the amount of disappearance at each pH with and without enzyme. Record your
results in Table 5 and on blackboard.
Table 5. Digestion of proteins by pepsin and trypsin.
Capillary
tube #
Solutions
1
pepsin in buffer, pH 2.0
2
pepsin in buffer, pH 4.0
3
pepsin in buffer, pH 6.0
4
pepsin in buffer, pH 8.0
5
buffer, pH 2.0
6
buffer, pH 4.0
7
buffer, pH 6.0
8
buffer, pH 8.0
9
trypsin in buffer, pH 2.0
10
trypsin in buffer, pH 4.0
11
trypsin in buffer, pH 6.0
12
trypsin in buffer, pH 8.0
Amount of egg white that
has disappeared (in %)
7
Why did we bother to incubate egg white in buffer solutions without enzymes?
How would you calculate the amount of egg white digested by pepsin and trypsin at different pH values?
CLEAN UP:

Wash all glassware with soap and warm water,
 Return equipment to where you found it.
NAME & section #:
Biology 153 LAB REPORT (2002-2003)
EXERCISE 7: DIGESTION.
INTRODUCTION
State the purpose of this exercise
MATERIALS AND METHODS
See the lab manual.
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RESULTS
I: DIGESTION OF FATS
1. Copy results in Table 2 to the table below.
Table A. Changes in colour observed during the digestion of milk fat by pancreatin.
Colors in test tubes during incubation
Test
tube #
0min
5min
10min
15min
30min
45min
1hr
1h15min
1h30min
1A
2A
3A
4A
2. Briefly describe the distribution of oil with and without bile salts (Exercise B).
II: DIGESTION OF CARBOHYDRATES
1. In the following table, copy results for the digestion of starch by amylase.
Table B. Benedict’s and iodine test results for starch digestion by amylase.
Test
tube #
pH
iodine test
colour
Benedict test
colour
Presence of
starch (yes/no)
1C
9
Presence of reducing
sugar (yes/no)
2C
3C
4C
III: DIGESTION OF PROTEINS
1. Complete the following table (Exercise D).
Table C. Digestion of egg white protein by pepsin and trypsin.
Capillary
tubes #
Solutions
1
pepsin in buffer, pH 2.0
2
pepsin in buffer, pH 4.0
3
pepsin in buffer, pH 6.0
4
pepsin in buffer, pH 8.0
5
buffer, pH 2.0
6
buffer, pH 4.0
7
buffer, pH 6.0
8
buffer, pH 8.0
9
trypsin in buffer, pH 2.0
10
trypsin in buffer, pH 4.0
11
trypsin in buffer, pH 6.0
12
trypsin in buffer, pH 8.0
Amount of egg
white that has
disappeared (in %)
Amount of egg white
that has been
digested (in %)
2. Prepare one graph showing the percent digestion of egg white by pepsin and trypsin.
Percent of digestion should be on the Y-axis and pH on the X-axis. Describe your results in no
more than 5 lines and explain the significance of the capillary tubes filled with egg white
incubated in buffers pH 2, 4, 6 and 8.
DISCUSSION
Answer in the spaces provided below. Do not use extra pages.
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I: DIGESTION OF FATS
Explain what happened in each tube used in Exercise A by completing the following table.
Test
tube #
Is the digestion of cream,
fast, slow or is there no
digestion at all?
Explain. Be sure to include: function of components of the test tube,
products of digestion, explanation for colour change, etc.
1A
2A
3A
4A
11
II: DIGESTION OF CARBOHYDRATES
Explain what happened in each tube used in Exercise C by completing the following table.
Test
tube #
Presence
of starch
(yes/no)
Presence of
reducing sugar
(yes/no)
Explain what is happening.
1C
2C
3C
4C
12
III:DIGESTION OF PROTEINS
Explain the results you graphed for the digestion of proteins (Exercise D).
Explain how optimum pH is created for these enzymes in the gastrointestinal tract (consider the
organs secreting these enzymes and the non-enzyme secretions at these locations).
13