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LAB EXERCISE
DIGESTION
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, explain how it is established, and state its
importance.
Explain the role of bile salts in digestion.
SAFETY (for Exercise C):
● Use extreme caution around hot plate. Wear Protective eyewear!!!! 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.
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I. DIGESTION OF FATS
A. THE DIGESTION OF MILK FATS BY PANCREATIC LIPASE
1. Label four test tubes with the sharpie and colored tape provided, #1A - 2A - 3A - 4A, and add solutions as
designated in Table 1. Add the enzyme last!
Table 1. Fat Digestion Tests with Pancreatic Lipase and Bile Salts
DISTILLED
WATER
ENZYME
(Pancreatic
Lipase)
Add last
Test
Tube
SUBSTRATE
(Cream)
BILE
SALTS
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
Notes:
i) Litmus is a pH indicator which changes color 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 color? If yes what is the color?
.
ii) Enzymatic digestion of fat (triglyceride) releases fatty acids that react with litmus and change its color to
red.
iii) Pancreatin is a commercial preparation of dried pancreatic enzymes.
2. Cover the tubes with parafilm and shake the tubes to mix thoroughly or use vortex mixer, make note of the
color of each tube, and place in a water bath at 37oC. Incubate for 1 to 1 1/2 hours.
3. Shake tubes and record their color in Table 2a every 10 minutes until no color change is observed
between color tests. At the end of the incubation period measure the pH of each tube by taking a
sample with disposable pipet and placing on probe sensor as shown in pictures. Don’t forget to rinse
the probe in the beaker marked “ph Rinse” and blot dry with Kimwipe.
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Choose terms to describe the colors such as: milky blue, milky violet, milky purple, pinkish purple,
reddish purple, transparent blue, transparent red… Write your results in Table 2a.
Table 2a. Color observations during digestion tests of milk fat by pancreatic lipase
Colors in test tubes during incubation
Test
tube
0min
10min
20min
30min
40min
50min
60min
70min
80min
90min
1A
2A
3A
4A
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4. Complete the table below:
Table 2b: Summary of results of milk fat digestion tests
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
3A
4A
B. DEMONSTRATION OF THE EMULSIFYING PROPERTIES OF BILE SALTS
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. Mix with vortex mixer. Then add 3.0 ml of oil.
2. When both test tubes are ready, mix using the vortex mixer. Try to mix one after another within 15
seconds. Put them back in your tray and keep both test tubes until the end of the laboratory period. Do
NOT shake them again. Describe the appearance of each test tube after first shaking and changes in
the appearance over time. Note generally how long it takes for the oil and water to separate.
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II. DIGESTION OF CARBOHYDRATES
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. Carbohydrate Digestion Tests with Pancreatic Amylase
Test
Tube
OTHER REAGENTS
SUBSTRATE
(starch solution)
ENZYME (Pancreatic
Amylase)
Add last
1C
5.O ml
---
5.0 ml
2C
5.0 ml
5.0 ml dist. Water
---
3C
---
5.0 ml dist. Water
5.0 ml
4C
5.0 ml
10 drops .2N HCl
5.0 ml
2. Shake the tubes and determine and record the pH of each reaction mixture in table 4. Since the pH probe
is too big for the mouth of the test tubes, pour the contents of each test tube into a clean 25 ml
beaker. The probe fits into these small beakers. After measuring the pH pour the contents of the
beaker back into the test tube for incubation. After each pH measurement, clean the probe according
to standard protocol.
3. Place the test tubes in a water bath at 37oC and incubate for at least 45 – 60 minutes. Shake the tubes every
15 minutes. Perform an iodine test and Benedict’s test at the end of the incubation period.
4. At the end of incubation, 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 color according to the
following scale:
Blue Green +
Yellow ++
Orange +++
Red ++++
Record the color of the precipitate in Table 4.
Perform the following control tests while waiting for the test tubes to incubate:
Lugol’s (IKI)
Iodine solution (Lugol’s) indicates the presence of starch by turning blue or black. Try it yourself!
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- 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 starch 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?
Keep these test tubes as controls to compare to the test tubes prepared for starch digestion.
Benedict’s
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. - 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 color 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 color of the precipitate for starch:
Record color of the precipitate for glucose:
Keep these test tubes as controls to compare to the test tubes prepared for starch digestion.
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Table 4. Results of iodine and Benedicts tests for starch digestion by amylase.
Test
tube
pH
Iodine test
(color)
Benedict
test
(color)
Presence
of starch
(yes/no)
Presence of
reducing sugar
(yes/no)
Brief explanation of each
result
1C
2C
3C
4C
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III. DIGESTION OF PROTEINS
EXERCISE D
Equipment:
● Egg white, scalpel, balance, weighing boats
(1)
pepsin in buffer, pH 2.0
(2)
pepsin in buffer, pH 8.0
(3)
trypsin in buffer, pH 2.0
(4)
trypsin in buffer, pH 8.0
and spatula
2% pepsin, 2g/100 ml water
2% pepsin, 2g/100 ml water
2% trypsin, 2 g/100 ml water
2% pepsin, 2g/100 ml water
1.
Obtain 4 test tubes and them as shown in table 5.
2.
Fill the test tubes with 10 ml of the solutions listed in table 5.
3.
Cut and weigh 4 small pieces of egg white and carefully add 1 piece to each tube using a small
spatula. Don’t forget to tare the balance with the weighing boat to make weighing easy.
4.
Record the weight that was used in each tube in table 5.
5.
Incubate at 37oC for 60 – 90 minutes. Try for 90 minutes
6.
Pour out most of the liquid from each tube.
7.
Remove each the egg white from each tube by tilting the tube and sliding the spatula under the
egg white.
8.
Remove most of the water by gently placing each side of the egg white on a paper towel, then
transfer to a tarred weighing boat and weigh. Record the weights in table 5.
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Table 5: Digestion of proteins by pepsin and trypsin.
Test
Tube
Solutions
1D
pepsin in buffer, pH 2.0
2D
pepsin in buffer, pH 8.0
3D
trypsin in buffer, pH 2.0
4D
trypsin in buffer, pH 8.0
Weight of egg
white at start
Weight of egg white
after incubation
Weight change
+/-
Why was the egg white in buffer solutions without enzymes?
Is there any way that the egg white could be transferred to improve the measurement at the end of the
incubation period?
CLEAN UP:
● Clean each table top with Lysol or Envirocide
● Return equipment to the location in the prep room. Ask the lab technician or teacher for the return location.
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LAB REPORT
EXERCISE DIGESTION.
INTRODUCTION
State the purpose of this exercise
MATERIALS AND METHODS
See the lab manual.
RESULTS
I: DIGESTION OF FATS
1. Copy results in Table 2a to the table below.
Table A. Changes in color observed during the digestion of milk fat by pancreatin.
Colors in test tubes during incubation
Test
tube
0min
10min
20min
30min
40min
50min
60min
70min
80min
90min
1A
2A
3A
4A
2. Briefly describe the distribution of oil with and without bile salts (Exercise B). Describe the appearance of
each test tube after first shaking and changes in the appearance over time. Note generally how long it takes for
the oil and water to separate.
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II: DIGESTION OF CARBOHYDRATES
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
(color)
Benedict test
(color)
Presence of starch
(yes/no)
Presence of reducing
sugar (yes/no)
1C
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 8.0
3
trypsin in buffer, pH 2.0
4
trypsin in buffer, pH 8.0
Weight of egg
white (+ or -)
Amount of egg
white that has
been digested (in
%)
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2. Prepare one graph showing the percent digestion of egg white by pepsin and trypsin in different buffers.
Percent of digestion should be on the Y-axis and pH on the X-axis. Paste or Insert graph below.
Describe your results and explain the significance of the tubes filled with egg white incubated in buffers pH 2,
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.
Table D: Discussion of results of milk fat digestion by pancreatic lipase
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 color change or no
color change, explanation for changes in rate of digestion, etc.
1A
2A
3A
4A
What is the significance of using this test tube?
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II: DIGESTION OF CARBOHYDRATES
Explain what happened in each tube used in Exercise C by completing the following table.
Table E: Discussion of results of carbohydrate digestion by pancreatic amylase
Test
tube
Presence
of starch
(yes/no)
Presence of
reducing sugar
(yes/no)
Explain your results.
1C
2C
3C
4C
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, the non-enzyme secretions at these locations, and the importance of pH in the activity
of each enzyme).
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Lab Materials
EXERCISE A
Equipment:
● bile salts (powder)
● pancreatin solution (2% - contains pancreatic lipase) 2g/100ml water, equally divided into 6 small clear
glass dropping bottles with caps.
● cream (half & half)
● litmus solution (1%) 1g/100ml water, equally divided into 6 small clear glass dropping bottles with caps.
● distilled water
● 5 ml pipettes
● pipette suction pump
● test tubes (20 x 100mm)
● water bath with test tube racks at 37 oC
● (6) 5ml, 2N HCl in small clear dropping bottle with attached eyedropper.
EXERCISE B
Equipment:
● bile salts (powder)
● distilled water
● olive oil
EXERCISE C
Equipment:
● starch solution (1%)
● distilled water
● .2N HCl (dropper bottles)
● pancreatin solution (2% - contains pancreatic amylase) 2g/100ml water
● test tubes
● paraffin to cover test tubes
● pH meter (Hach probe type)
● water bath at 37oC
● iodine solution (Lugol’s)
● glucose solution (1%) - 1g/100 ml water
● Benedict's solution
● hot plate
● test tube clamps
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