3.3 ACTION OF DIGESTIVE ENZYMES
Time required: 2 double periods
Name:_________________
Introduction: Digestive enzymes are found in our digestive system where they break down macromolecules
in the food into their smaller building blocks. In humans digestion takes place in the mouth, stomach and small
intestines. The digestive enzymes are secreted by different glands located in these organs. Polysaccharides in
starch are converted by salivary amylase and pancreatic amylase into maltose which is further converted to
glucose by maltase in intestinal cells. Pepsin from stomach and trypsin from pancreas convert proteins to
peptides which in turn are converted to peptides by peptidases from intestinal cells. Lipase produced by
pancreas breaks down lipids into fatty acids and glycerol. In this experiment you will investigate the action of
salivary amylase on starch, pepsin on egg albumin (protein) and the action of lipase on milk fat.
Learning outcomes: After completing this investigation, students will be able to understand how salivary
amylase, pepsin and lipase break down the macromolecules in starch, protein and fat into their monomers.
Group size: 6 groups each with 3-4 students
Pre-lab activity: Read instructions for this investigation prior to the lab and define the key words.
Key terms: Digestion, digestive enzyme, polysaccharide, amylase, protease, lipase and maltase.
Suggestion for teachers: Divide the class into 6 groups. Parts A, B, and C can be carried out by 2 groups
each and pool their results.
Part A- action of salivary amylase on starch
Salivary amylase digests starch to maltose by hydrolysis. When starch solution is tested with iodine solution
before digestion in turns blue black. After digestion the maltose formed reacts with Benedict’s solution and
turns orange in colour. Starch + Amylase
Maltose
Objective: To investigate the action of salivary amylase on starch.
Hypothesis: Write a hypothesis about the action of salivary amylase on starch.
Salivary amylase converts starch to maltose.
Apparatus and materials per group:
Test tubes -8
Test tube rack -1
Test tube holder-1
Water proof pen
Salivary amylase
Graduated pipette-10 mL
500 mL beaker-2
Bunsen burner
Safety goggles
Glass rod
Tripod stand
Gauze mat
Heat mat
Matches
2% starch solution
Lugol’s iodine solution
Benedict’s solution
Distilled water`
1% sodium hypochlorite In a beaker
Figure 1- Experimental set up for amylase and starch reaction
Test tube 1
Test tube 2
Test tube 3
Test tube 4
Test tube 5
Test tube 6
Test tube 7
Test tube 8
Boiled
amylase
Starch
solution
+ boiled
amylase
Add
Benedict’s
solution
and heat
Starch
solution
+ unboiled
amylase
Add
Benedict’s
solution
and heat
Starch
solution
+ boiled
amylase
Add iodine
solution
Starch
solution
+ unboiled
amylase
Add iodine
solution
Starch
solution
Control 1
Starch
solution
Control 2
Add iodine
solution
Add
Benedict’s
Solution
and heat
Unboiled
amylase
Test with
1
Method:
1. Prepare a water bath by using a Bunsen burner to heat some water in a beaker until it boils; then turn the
flame down to keep the water just boiling.
2. Label eight test tubes 1 to 8 with a water proof pen as shown in figure 1.
3. Pour 25 mL amylase enzyme in test tube 1 and test tube 2.
4. Place test tube in the boiling water bath for 3 minutes.
Note: When amylase is boiled, it denatures and does not react with starch.
5. Using a graduated pipette add 5 mL of starch solution to test tubes 3 to 8.
6. Using a new pipette transfer 5 mL boiled amylase from test tube 2 to test tubes 3 and 5. Stir the contents
using a glass rod.
7. Add 2 to 3 drops of iodine solution to test tubes 5, 6 and 7 and note any colour changes.
Note: The solution turns blue black if starch is present. If starch is converted to maltose by amylase, the
solution will not turn blue black,
8. Add 5 mL Benedict’s solution to test tube 3, 4 and 8. Place these three test tubes in a hot water bath for 5
minutes and note any colour changes.
Note: The solution turns orange in colour if starch is converted to maltose by amylase. If starch is not
digested, the colour will not change.
13. Record your results in data table 1.
Results: Table 1- Benedict’s test and iodine test results
Test
Tube
Contents
Tested with
3
Starch and boiled amylase Benedict’s solution
4
Starch and amylase
5
Starch and boiled amylase Iodine solution
6
Starch and amylase
7
Starch solution (control 1) Iodine solution
8
Starch solution (control 2) Benedict’s solution
Benedict’s solution
Iodine solution
Result
Change in colour
No change
Turns orange
Turns blue black
No change
Turns blue black
No change
Interpretation
Boiling denatured
amylase
Starch is digested to
maltose by amylase
Boiling denatured
amylase
Starch is digested to
maltose by amylase
Starch reacted with
iodine
In the absence of
amylase starch was not
digested to maltose.
Discussion questions:
1. Which substances do Iodine and Benedict’s solution test for?
Iodine is used to test for starch and Benedict’s solution is used to test for glucose.
2. Did you observe any changes in colour in test tubes 3, 4 and 8 tested with Benedict’s solution? If so
what substance was present in these 3 test tubes?
Test tube3: starch and boiled saliva+ Benedict’s solution- No colour change because when amylase
was boiled it denatured and did not react with starch.
Test tube 4: Starch and saliva+ Benedict’s solution- Colour changed to orange because amylase
digested starch to maltose.
Test tube 8: Starch + Benedict’s solution- No colour change because due to the absence of amylase,
starch was not digested.
3. Did you observe any changes in colour in test tubes 5, 6 and 7 tested with iodine solution? If so what
substance was present in these 3 test tubes?
Test tube 5: Starch and boiled saliva + Iodine- colour changed to blue black because starch was not
digested by denatured amylase.
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Test tube 6: Starch and saliva + Iodine- no colour change because starch is digested to maltose
Test tube 7: Starch + Iodine- Colour changes to blue black because iodine reacts with starch.
4. What change takes place when starch and saliva are mixed, according to the results in tubes 4 and 6?
In test tube 4 starch was digested by salivary amylase to maltose. It reacted with Benedict’s solution
and turned orange in colour.
In test tube 6 also starch was digested by salivary amylase to maltose. Maltose did not react with
iodine.
5. Do your results prove that amylase reacts with starch and changes it into maltose?
My results proved that amylase has changed starch to maltose.
6. Did your results support your hypothesis? Yes. My results supported my hypothesis that amylase
digests starch to maltose.
Conclusion: Explain what happens when salivary amylase is added to starch solution.
When salivary amylase is added to starch which is a polysaccharide, it converts starch to a disaccharide
(maltose) by hydrolysis (by removin water)
Part B -Action of pepsin on egg white protein
Egg-white suspension consists of solid particles of albumin (protein), suspended in water and the solution will
be cloudy in appearance. In acidic medium (in the presence of HCl), pepsin breaks down protein into peptides.
After the protein in egg white is digested by pepsin enzyme, the solution becomes clear because peptides are
soluble in water.
Protein + pepsin
peptides
Objective: To investigate the action of pepsin on egg white protein.
Hypothesis: Write a hypothesis about the action of pepsin on egg white protein.
Pepsin digests protein in egg white into peptides by hydrolysis.
Apparatus and materials per group:
Test tubes- 4
Test tube rack-1
Test tube holder-1
250 ml beaker-1
Thermometer -1
Dropping pipette-1
Graduated pipette-10cm3
Water proof pen
Lab coat
Bunsen burner
Tripod stand
Gauze mat
Heat mat
Goggles
Tongs-1
Matches
1% egg-white suspension
1% pepsin solution
2M hydrochloric acid
Figure 2- Experimental set up for pepsin and egg white reaction
Test tube 1
Test tube 2
Test tube 3
Test tube 4
Egg white suspension
+ Pepsin
Control
Egg white suspension
+ hydrochloric acid
No pepsin
Egg white suspension
+ hydrochloric acid
+ Pepsin
Egg white suspension
+ hydrochloric acid
+ boiled Pepsin
Method:
Note: Egg-white suspension looks cloudy because it consists of solid particles of albumin, suspended in
water. The pH of stomach is acidic because hydrochloric acid is produced. Enzymes are denatured when
boiled.
1. Label four test-tubes 1-4 as shown in table 3.
2. Add 10 mL egg white suspension to all 4 test tubes.
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3. Add 5 mL of dilute hydrochloric acid to tubes 2, 3 and 4.
4. Using a graduated pipette place 1 mL pepsin solution in the 5th test tube and heat it over a small Bunsen
flame until the liquid boils. Add the boiled pepsin to the egg-white suspension in test tube 4.
Caution: Be careful when using protein and hydrochloric acid. Take care when handling hot plate and
hot glass ware.
5. Prepare a water bath in a 250 mL beaker and maintain the temperature of water at about 40 °C.
6. Using a graduated pipette, add 1mL pepsin to tubes 1 and 3 only.
7. Place all four tubes in the water bath for 6 minutes.
8. After 6 minutes remove the four test tubes from the water bath and place them in the test-tube rack.
9. Note the appearance of the egg white suspension in each test tube and record your results in table 4.
Results: Table 2- Action of pepsin on egg white suspension
Test tube
1
Control
Contents
Appearance of egg
white suspension
Egg-white suspension & pepsin
Cloudy
2
Egg-white suspension & HCl
Cloudy
3
Egg-white suspension, pepsin & HCl
4
Egg-white suspension, boiled pepsin & HCl Cloudy
Clear
Inference
Protein is not digested
because HCl was not
added
Protein is not digested
Because pepsin was not
added
Protein is digested
Protein is not digested
because boiled pepsin
was denatured
Discussion questions:
1. Why does the egg white suspension look cloudy?
Egg white suspension looks cloudy because it contains solid particles of albumin suspended in water.
2. If the egg-white suspension goes from cloudy to clear, what change must have occurred?
When egg-white is digested by pepsin, it changes to peptides which are soluble in water
3. What was the reason for adding hydrochloric acid to test tubes 2, 3 and 4?
Pepsin breaks down protein only when the pH is acidic. That is why HCl is added to test tubes 2, 3 and 4.
4. In which of the test-tubes are the conditions most like those in the stomach?
In test tube 3, the conditions are most like those in the stomach because it contained hydrochloric acid and
provided acidic medium for pepsin to react with the egg white.
5. What happened when pepsin was boiled? When pepsin was boiled it denatured and could not react with the
egg white,
6. What was the purpose of test tube 1? Test tube 1 is a control test tube.
7. Do the results with test tube 3 prove that pepsin is an enzyme that converts proteins to peptides?
The results with the test tube 3 prove that pepsin is an enzyme that converts proteins to peptides in acidic
medium.
8. Did your test results support your hypothesis? Yes. My results supported my hypothesis that pepsin converts
protein in egg white to peptides.
Conclusion: Explain what happens when pepsin and hydrochloric acid were added to egg white suspension.
When pepsin and hydrochloric acid were added to egg white suspension, the protein in egg white was
digested to peptides by hydrolysis.
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Part C- Action of lipase on milk fat
Milk contains protein (caseinogen), fat (butter fat) and sugar (lactose). Bile salts turn the solution alkaline.
When the milk fat is digested by lipase into fatty acids and glycerol the solution becomes acidic.
Phenolphthalein is a pH indicator. In alkaline solutions (above pH 7) it is pink; in acidic solutions (below pH 7)
it is colourless. Milk fat + lipase
fatty acids+ glycerol
Objective: To investigate the action of lipase on milk fat.
Hypothesis: Write a hypothesis about the action of lipase on milk fat
Lipase enzyme will digest milk fat into fatty acids and glycerol.
Apparatus and materials per group:
Test tubes -4
Test tube rack -1
Test tube holder-1
Water proof pen
Clock
Graduated pipette-10cm3
500 mL beaker-2
Bunsen burner
Safety goggles
Dropping pipette
Tripod stand
Gauze mat
Heat mat
Matches
Lab coat
Milk
3% bile salts solution
Sodium carbonate solution
Phenolphthalein indicator
5 % lipase solution
Figure 3- Experimental set up for action of lipase on milk fat
Test tube 1
Milk, sodium carbonate
and phenolphthalein +
unboiled lipase
Control
Test tube 2
Milk, sodium carbonate and
phenolphthalein+ boiled lipase
+ bile salts
Test tube 3
Milk, sodium carbonate and
phenolphthalein+ lipase
+ bile salts
Test tube 4
Milk, sodium
carbonate and
phenolphthalein
+boiled lipase
Note: Sodium carbonate is added to make the solution alkaline. Lipase needs alkaline medium to react with
the milk fat. Bile salts break the fat molecules smaller by emulsification so that the enzyme can digest fat
molecules faster.
Method:
1. Label three test-tubes as shown in figure 3.
2. Using graduated pipette place 10 mL milk in each tube.
3. Rinse the pipette and place 5 mL dilute sodium carbonate solution in each test tube to make the mixture
alkaline.
4. Rinse the pipette and place 2 mL bile salts solution in test tubes 2 and 3 only.
5. Use a dropping pipette add 6 drops of phenolphthalein solution to each test tube.
Note: Phenolphthalein turns pink in alkaline solution and colourless in acidic solutions.
6. In the 4th test tube, place about 1) mL of lipase solution and heat the liquid over a small Bunsen flame until it
boils for a few seconds.
7. Cool the tube under the tap and, transfer 2 mL of the boiled lipase to tube 2.
8. With the graduated pipette place 2mL unboiled lipase solution in test tubes 1 and 3.
9. Mix the contents of each test tube with separate glass rods and place them in a test tube rack.
10. Observe the change in the colour of the phenolphthalein indicator.
11. Note the time required for the contents of each tube to go clear and then enter your results in table 3.
Note: If the medium of the solution becomes acidic phenolphthalein turns clear. When milk fat is digested by
lipase into fatty acids and glycerol, the medium turns acidic.
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Results: Table 3- Action of lipase on milk fat
Test
tube
1
2
Colour change
of
phenolphthalein
Contents
Milk+ sodium carbonate
+ phenolphthalein + lipase (control)
Milk+ sodium carbonate
+ phenolphthalein + boiled lipase
+ bile salts
Pink
Pink
3
Milk+ sodium carbonate
+ phenolphthalein + lipase + bile salts
4
Milk + sodium carbonate
+ phenolphthalein +boiled lipase
Clear
Pink
Inference
Milk fat is not digested because bile
salts were not added
Milk fat is not digested because
lipase was denatured by boiling
Milk fat was digested to fatty acids
and glycerol and the solution
became acidic
Milk fat is not digested because
lipase was denatured by boiling.
Discussion questions:
1 What food substances are present in milk?
Milk contains protein (caseinogen), fat (butter fat) and sugar (lactose).
2. What was the purpose of using test tube 1? Test tube 1 is used as a control.
3. In which test tube did the colour of phenolphthalein change?
In test tube 3 the colour of phenolphthalein changed from pink to clear because fat was digested to fatty
acids and glycerol by the action of lipase.
4. What can you infer bout this change in colour?
The colour of phenolphthalein changed from pink to clear because fat in milk was digested by the enzyme
lipase into fatty acids and glycerol which made the solution acidic.
5. What is the action of lipase on milk fat? Lipase digested milk fat into fatty acid and glycerol.
6. Which part of the experiment suggested that lipase acts as an enzyme?
Reaction in test tube 3 suggested that lipase acts as an enzyme because milk fat was broken down to fatty acid
and glycerol.
8 From the results, assuming that lipase is an enzyme, what part do the bile salts appear to be playing in the
reaction? Bile salts emulsify the fat into smaller molecules enabling lipase to react with these fat molecules.
9. Did your test results support your hypothesis? Yes. My results supported my hypothesis that lipase digests
milk fat into fatty acid and glycerol.
Conclusion: Explain what happens when lipase is added to milk.
When lipase is added to milk, the fat in milk is converted to fatty acid and glycerol in the presence of bile
salts and sodium carbonate
Web sites:
http://www.accessexcellence.org/AE/ATG/data/released/0334-RobertGoodman/index.php
http://www.biotopics.co.uk/nutrition/amylex.html
http://www.biotopics.co.uk/nutrition/pepsin.html
http://www.biotopics.co.uk/nutrition/lipase.html
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